DNA encoding a human 5-HT1F receptor and uses thereof

ABSTRACT

This invention provides an isolated nucleic acid molecule encoding a human 5-HT 1F  receptor, an isolated protein which is a human 5-HT 1F  receptor, vectors comprising an isolated nucleic acid molecule encoding a human 5-HT 1F  receptors. mammalian cells comprising such vectors, antibodies directed to the human 5-HT 1F  receptor, nucleic acid probes useful for detecting nucleic acid encoding human 5-HT 1F  receptors, antisense oligonucleotides complementary to any sequences of a nucleic acid molecule which encodes a human 5-HT 1F  receptor, pharmaceutical compounds related to human 5-HT 1F  receptors, and nonhuman transgenic animals which express DNA a normal or a mutant human 5-HT 1F  receptor. This invention further provides methods for determining ligand binding, detecting expression, drug screening, and treatment involving the human 5-HT 1F  receptor.

BACKGROUND OF THE INVENTION

[0001] Throughout this application various publications are referencedby partial citations within parentheses. The disclosures of thesepublications in their entireties are hereby incorporated by reference inthis application in order to more fully describe the state of the art towhich this invention pertains.

[0002] Since the purification of a pressor substance in blood serumtermed serotonin (Rapport et al., 1947) and later identified as5-hydroxytryptamine (5-HT) (Rapport, 1949), there has been a plethora ofreports demonstrating that this indoleamine not only plays a role in thefunctioning of peripheral tissues but, indeed, performs a key role Inthe brain as a neurotransmitter. Certainly, the anatomical localizationof serotonin and serotonergic neurons in both the peripheral and centralnervous systems supports its role in such diverse physiologic andbehavioral functions as pain perception, sleep, aggression, sexualactivity, hormone secretion, thermoregulation, motor activity,cardiovascular function, food intake and renal regulation (For reviewsee Green, 1985; Osborne and Hamon, 1988; Sanders-Bush, 1988; Peroutka,1991;. Taken together, it appears that serotonin plays an important rolein homeostasis and in modulating responsiveness to environmentalstimuli. Accordingly, studies demonstrating that abnormalities in theserotonergic system may be associated with disease states has created adrug development effort towards agents which may selectively modulatethe function of serotonin (Glennon, 1990).

[0003] In relation to the characterization of physiologic or biochemicalresponses resulting from the release of serotonin are simultaneousinvestigations examining the receptor sites responsible for the actionselicited by the indoleamine transmitter. Following early in vitropharmacological assays describing the existence of two differentserotonin receptors, designated as D and M, in the guinea pig ileum(Gaddum and Picarelli, 1957), the advent of receptor binding techniquein the 1970's has brought to light during the last decade the diversityof 5-HT receptors existing in both the brain and peripheral tissues.Thus, although the concept of D and M receptors has not beeninvalidated, serotonin receptors not fitting either category have beenidentified using radioligand methods. To date using this technique,there appears to be four classes of serotonin receptors found in thebrain: 5-HT₁, 5-HT₂, 5-HT₃ and, putatively, 5-HT₄ (Peroutka, 1991).Furthermore, 5-H₁ sites have been, subclassified as: 5-HT_(1A),5-HT_(1B), 5-HT_(1C), 5-HT_(1D) (Hamon et al., 1990) and 5-HT_(1E)(Leonhardt et al., 1989). Although a detailed characterization of the5-HT_(1F) binding site is lacking, extensive pharmacologic, biochemicaland functional properties have clearly shown that the other foursubtypes of 5-HT₁ sites are receptors according to classical criteria.

[0004] During the last few years, the field of molecular biology hasprovided an important facet to receptor research by cloning theseproteins and allowing more precise characterizations in isolated systems(Hartig et al,1990). This has seen accomplished for the 5-H_(1A) (Farginet al., 1988), 5-HT_(1C) (Julius et al., 1988), 5-HT_(1D) (Branchek etal., 1990) and 5-HT₂ receptors (Pritchett et al., 1988). Thus, there isno doubt that these binding sites represent “true” functional receptors.Indeed, the pharmacological characterization of serotonin receptorsinvolved in various physiological or biochemical functions is a keycomponent of drug development for the serotonergic system. As one candeduce from the diversity of serotonin binding sites, many targets areavailable for advancement in selective drug design. The coupling ofmolecular biological methods to pharmacological characterizationparticularly for cloned human receptors will open new avenues forpharmaceutical development which has not been previously explored.

[0005] This study is a pharmacological characterization of aserotonergic receptor clone with a binding profile different from thatof any serotonergic receptor to date. In keeping with the nomenclaturepresently accepted for serotonin receptors, this novel site will betermed a 5-HT_(1F) receptor based upon the fact that it possesses highaffinity for the endogenous neurotransmitter, 5-HT.

SUMMARY OF THE INVENTION

[0006] This invention provides an isolated nucleic acid moleculeencoding a human 5-HT_(1F) receptor (Seq. I.D. No. 1).

[0007] This invention also provides an isolated protein which is a human5-HT_(1F) receptor (Seq. I.D. Nos. 2, 7).

[0008] This invention provides a vector comprising an isolated nucleicacid molecule encoding a human 5-HT_(1F) receptor.

[0009] This invention also provides vectors such as plasmids comprisinga DNA molecule encoding a human 5-HT_(1F) receptor, adapted forexpression in a bacterial cell, a yeast cell, or a mammalian cell whichadditionally comprise the regulatory elements necessary for expressionof the DNA in the bacterial, yeast, or mammalian cells so locatedrelative to the DNA encoding the 5-HT_(1F) receptor as to permitexpression thereof.

[0010] This invention provides a mammalian cell comprising a DNAmolecule encoding a human 5-HT_(1F) receptor.

[0011] This invention provides a method for determining whether a ligandnot known to be capable of binding to a human 5-HT_(1F) receptor canbind to a human 5-HT_(1F) receptor which comprises contacting amammalian cell comprising an isolated DNA molecule encoding a human5-HT_(1F) receptor with the ligand under conditions permitting bindingof ligands known to bind to a 5-HT_(1F) receptor, detecting the presenceof any of the ligand bound to a human 5-HT_(1F) receptor, and therebydetermining whether the ligand binds to a human 5-HT_(1F) receptor.

[0012] This invention also provides a method for determining whether aligand not known to be capable of binding to the human 5-HT_(1F)receptor can functionally activate its activity or prevent the action ofa ligand which does so This comprises contacting a mammalian cellcomprising an isolated DNA molecule which encodes a human 5-HT_(1F)receptor with the ligand under conditions permitting the activation orblockade of a functional response, detected by means of a bioassay fromthe mammalian cell such as second messenger response, and therebydetermining whether the ligand activates or prevents the activation ofthe human 5-HT_(1F) receptor functional output.

[0013] This invention further provides a method of screening drugs toidentify drugs which specifically interact with, and bind to, the human5-HT_(1F) receptor on the surface of a cell which comprises contacting amammalian cell comprising an isolated DNA molecule encoding a human5-HT_(1F) receptor with a plurality of drugs, determining those drugswhich bind to the mammalian cell, and thereby identifying drugs whichspecifically interact with, and bind to, a human 5-HT_(1F) receptor.

[0014] This invention also provides a method of screening drugs toidentify drugs which interact with, and activate or block the activationof, the human 5-HT_(1F) receptor on the surface of a cell whichcomprises contacting the mammalian cell comprising an isolated DNAmolecule encoding and expressing a human 5-HT_(1F) receptor with aplurality of drugs, determining those drugs which activate or block theactivation of the receptor in the mammalian cell using a bioassay suchas a second messenger assays, and thereby identifying drugs whichspecifically interact with, and activate or block the activation of, ahuman 5-HT_(1F) receptor.

[0015] This invention provides a nucleic acid probe comprising a nucleicacid molecule of at least 15 nucleotides capable of specificallyhybridizing with a sequence included within the sequence of a nucleicacid molecule encoding a human 5-HT_(1F) receptor.

[0016] This invention also provides a method of detecting expression ofthe 5-HT_(1F) receptor on the surface of a cell by detecting thepresence of mRNA coding for a 5-HT_(1F) receptor which comprisesobtaining total mRNA from the cell and contacting the mRNA so obtainedwith a nucleic acid probe comprising a nucleic acid molecule of at leas15 nucleotides capable of specifically hybridizing with a sequenceincluded within the sequence of a nucleic acid molecule encoding a human5-HT_(1F) receptor under hybridizing conditions, detecting the presenceof mRNA hybridized to the probe, and thereby detecting the expression ofthe 5-HT_(1F) receptor by the cell.

[0017] This invention provides an antisense oligonucleotide having asequence capable of binding specifically with any sequences of an mRNAmolecule which encodes a human 5-HT_(1F) receptor so as to preventtranslation of the mRNA molecule.

[0018] This invention provides an antibody directed to a human 5-HT_(1F)receptor.

[0019] This invention provides a transgenic nonhuman mammal expressingDNA encoding a human 5-HT_(1F) receptor. This invention also provides atransgenic nonhuman mammal expressing DNA encoding a human 5-HT_(1F)receptor so mutated as to be incapable of normal receptor activity, andnot expressing native 5-HT_(1F) receptor. This invention furtherprovides a transgenic nonhuman mammal whose genome comprises antisenseDNA complementary to DNA encoding a human 5-HT_(1F) receptor so placedas to be transcribed into antisense mRNA which is complementary to mRNAencoding a 5-HT_(1F) receptor and which hybridizes to mRNA encoding a5-HT_(1F) receptor thereby reducing its translation.

[0020] This invention provides a method of determining the physiologicaleffects of expressing varying levels of human 5-HT_(1F) receptors whichcomprises producing a transgenic nonhuman animal whose levels of human5-HT_(1F) receptor expression are varied by use of an inducible promoterwhich regulates human 5-HT_(1F) receptor expression.

[0021] This invention also provides a method of determining thephysiological effects of expressing varying levels of human 5-HT_(1F)receptors which comprises producing a panel of transgenic nonhumananimals each expressing a different amount of human 5-HT_(1F) receptor.

[0022] This invention provides a method for diagnosing a 15predisposition to a disorder associated with the expression of aspecific human 5-HT_(1F) receptor allele which comprises: a. obtainingDNA of subjects suffering from the disorder; b. performing a restrictiondigest of the DNA with a panel of restriction enzymes; c.electrophoretically separating the resulting DNA fragments on a sizinggel; d. contacting the resulting gel with a nucleic acid probe capableof specifically hybridizing to DNA encoding a human 5-HT_(1F) receptorand labelled with a detectable marker; e. detecting labelled bands whichhave hybridized to the DNA encoding a human 5-HT_(1F) receptor labelledwith a detectable marker to create a unique band pattern specific to theDNA of subjects suffering from the disorder; f. preparing DNA obtainedfor diagnosis by steps a-e; and g. comparing the unique band patternspecific to the DNA of subjects suffering from the disorder from step eand the DNA obtained for diagnosis from step f to determine whether thepatterns are the sane or different and to diagnose therebypredisposition to the disorder if the patterns are the same.

[0023] This invention provides a method of preparing the isolated5-HT_(1F) receptor which comprises inducing cells to express 5-HT_(1F)receptor, recovering the receptor from the resulting cells and purifyingthe receptor so recovered.

[0024] This invention also provides a method of preparing the isolated5-HT_(1F) receptor which comprises inserting nucleic acid encoding5HT_(1F) receptor in a suitable vector, inserting the resulting vectorin a suitable host cell, recovering the receptor produced by theresulting cell, and purifying the receptor so recovered.

[0025] This invention provides an antisense oligonucleotide having asequence capable of binding specifically with any sequences of an mRNAmolecule which encodes a receptor so as to prevent translation of themRNA molecule.

[0026] This invention also provides a transgenic nonhuman mammalexpressing DNA encoding a receptor.

[0027] This invention further provides a transgenic nonhuman mammalexpressing DNA encoding a receptor so mutated as to be incapable ofnormal receptor activity, and not expressing native receptor.

[0028] This invention also provides a method of determining thephysiological effects of expressing varying levels of a receptor whichcomprises producing a transgenic nonhuman animal whose levels ofreceptor expression are varied by use of an inducible promoter whichregulates receptor expression.

[0029] This invention also provides a method of determining thephysiological effects of expressing varying levels of a receptor whichcomprises producing a panel of transgenic nonhuman animals eachexpressing a different amount of the receptor.

[0030] This invention further provides a transgenic nonhuman mammalwhose genome comprises antisense DNA complementary to DNA encoding areceptor so placed as to be transcribed into antisense mRNA which iscomplementary to mRNA encoding the receptor and which hybridizes of mRNAencoding the receptor thereby preventing its translation.

[0031] This invention provides a method for determining whether a ligandnot known to De capable of binding to a receptor can bind to a receptorwhich comprises contacting a mammalian cell comprising an isolated DNAmolecule encoding the receptor with the ligand under conditionspermitting binding of ligands known to bind to a receptor, detecting thepresence of any of the ligand bound to the receptor, and therebydetermining whether the ligand binds to the receptor.

BRIEF DESCRIPTION OF THE FIGURES

[0032]FIG. 1. (FIGS. 1A-1F) Nucleotide and deduced amino acid sequenceof gene 5-HT_(1F) (Seq. I.D. Nos. 1, 2, and 7).

[0033] Numbers above the nucleotide sequence indicate nucleotideposition. DNA sequence was determined by the chain termination method ofSanger, et al., on denatured double-stranded plasmid templates using theenzyme Sequenase. Deduced amino acid sequence (single letter code) of along open reading frame is shown.

[0034]FIG. 2. (FIGS. 2A-2D) Comparison of the human 5-HT_(1F) receptorprimary structures with other serotonin is: receptors (Seq. I.D. Nos.:5-HT_(1A)—3; 5-HT_(1C)—4; 5-HT_(1Dα)—5; 5-HT_(1Dβ)—6; 5-HT_(1F)—7;5-HT₂—8).

[0035] Amino acid sequences (single letter code) are aligned to optimizehomology. The putative transmembrane spanning domains are indicated bystars and identified by Roman numerals (TM I-VII).

[0036]FIG. 3. 5-HT concentration-effect curves are represented in theabsence () and in the presence (o) of methiothepin (1.0 μM). Data werenormalized to 100% relative to forskolin-stimulated values in theabsence of agonist to derive values of E_(max) and E₅₀. The antagonistK_(b) was estimated by method of Furchgott (32): K_(b)=(Dose ofantagonist)/((E₅₀ in the presence of antagonist/control E₅₀)−1).

[0037]FIG. 4. Human tissue distribution of RNA coding for 5-HT_(1F)receptor gene. Total RNA was converted to single-stranded cDNA byrandom-priming with reverse transcriptase. cDNAs were amplified by PCRusing 5-HT_(1F) specific PCR primers. PCR products were run on a 1.5%agarose gel, blotted onto nylon membranes and hybridized to internalgene-specific oligonucleotides and washed under high stringency.Positive controls represent gene-specific recombinant plasmids; dH₂Oserved as a negative control. PCR amplification and Southern blotting ofRNA samples not treated with reverse transcriptase were negative.

[0038]FIG. 5: 5-HT_(1F) receptor mRNA in the guinea pig brain coronalsections. A. An antisense oligonucleotide probe (4,5 loop) was used. Anidentical pattern was observed with the 5′ untranslated probe (notillustrated. Hybridization densities are high in layer V of cerebralcortex (V), and in CA1-CA3 of the hippocampus (HC). B. Controlcontralateral hemisphere of an adjacent section to that in A. Nohybridization was seen using a sense probe of identical specificactivity. C. Section hybridized with the antisense probe. The dorsalraphe (DR) is densely labeled. D. At high magnification, hybridization(antisense probe) is detected in layer V of sensorimotor cortex.Arrowheads indicate heavily labeled pyramidal cells. E. As in D, throughthe dorsal raphe. Arrowheads indicate large, heavily labeled neurons.Magnification in panels D and E=X270.

DETAILED DESCRIPTION OF THE INVENTION

[0039] As used herein, the 5-HT receptor family is defined as the groupof mammalian proteins that function as receptors for serotonin. A 5-HTreceptor subfamily is defined as a subset of proteins belonging to the5-HT receptor family which are encoded by genes which exhibit homologyof greater than 72% or higher with each other in their deduced aminoacid sequences within presumed transmembrane regions (linearlycontiguous stretches of hydrophobic amino acids, bordered by charged orpolar amino acids, that are long enough to form secondary proteinstructures that span a lipid bilayer). Four-human 5-HT receptorsubfamilies can be distinguished based on the information presentlyavailable: 5-H₁, 5-HT₂, 5-HT₃ and 5-HT₄ (Peroutka, 1991). The 5-HT₂receptor subfamily contains the human 5-HT₂ receptor. Although no otherhuman members of this family have been described, the rat 5-HT₂ receptor(Pritchett, et al. 1988; Julius, et al. Proc. Natl. Acad. SCi. USA 8:928-932, 1990) and the rat 5HT_(1C) receptor (Jullus, et al. 1988)constitute a rat 5-HT receptor subfamily. The 5-HT, subfamily has beensubdivided further as: 5-HT_(1A), 5-HT_(1B), 5-HT_(1C), 5-HT_(1D) (Hamonet al., 1990) and 5-HT_(1E) (Leonhardt et al., 1989). The 5-HT_(1A)subfamily contains the human 5-HT_(1A) receptor, also known as G-21(Fargin, et al. 1988) The 5-HT_(1D) receptor subfamily contains twomembers, the 5-HT_(1D-1) receptor (also termed 5-HT_(1Dα)) and the5-HT_(1D-2) receptor (also termed 5-HT_(1Dβ)) The 5-HT_(1F) subfamilycontains the human 5-HT_(1F) receptor (also termed clone h116a).Although this definition differs from the pharmacological definitionused earlier, there is significant overlap between the presentdefinition and the pharmacological definition. Members of the 5-HT_(1F)receptor subfamily so described include the 5-HT_(1F) receptor and an)other receptors which have a greater than 72% homology to the DNA andamino acid sequence shown in FIG. 1 (Seq. I.D. Nos. 1, 2, and 7)according to the definition of “subfamily”. This invention relates tothe discovery of the first member of the human 5-HT_(1F) receptorsubfamily.

[0040] This invention provides an isolated nucleic acid moleculeencoding a human 5-HT_(1F) receptor (Seq. I.D. No. 1). As used herein,the term “isolated nucleic acid molecule” means a nucleic acid moleculethat is, a molecule in a form which does not occur in nature. Such areceptor is by definition a member of the 5-HT_(1F) receptor subfamily.Therefore, any receptor which meets the defining criteria given above isa human 5-HT_(1F) receptor. One means of isolating a human 5-HT_(1F)receptor is to probe a human genomic library with a natural orartificially designed DNA probe, using methods well known in the art.DNA probes derived from the human receptor gene 5-HT_(1F) areparticularly useful probes for this purpose. DNA and cDNA moleculeswhich encode human 5-HT_(1F) receptors may be used to obtaincomplementary genomic DNA, cDNA or RNA from human, mammalian or otheranimal sources, or to isolate related cDNA or genomic clones by thescreening of cDNA or genome libraries, by methods described in moredetail below, transcriptional regulatory elements from the 5′untranslated region of the isolated clones, and other stability,processing, transcription, translation, and tissuespecificity-determining regions from the 3′ and 5′ untranslated regionsof the isolated genes are thereby obtained. Examples of a nucleic acidmolecule are an RNA, cDNA, or isolated genomic DNA molecule encoding ahuman 5-HT_(1F) receptor. Such molecules may have coding sequencessubstantially the same as the coding sequence shown in FIG. 1. The DNAmolecule of FIG. 1 encodes the sequence of the human 5-HT_(1F) receptorgene (Seq. I.D. No. 1).

[0041] This invention further provides a cDNA molecule of encoding ahuman 5-HT_(1F) receptor having a coding sequence substantially the sameas the coding sequence shown in FIG. 1 (Seq. I.D. No. 1). This moleculeis obtained by the means described above.

[0042] This invention also provides an isolated protein which is a human5-HT_(1F) receptor. As used herein, the tern. “isolated protein means aprotein molecule free of other cellular components. An example of suchprotein is an isolated protein having substantially the same amino acidsequence as the amino acid sequence shown in FIG. 1 (Seq. I.D. Nos. 2,7) which is a human 5-HT_(1F) receptor. One means for obtaining isolated5-HT_(1F) receptor is to express DNA encoding the receptor in a suitablehost, such as a bacterial, yeast, or mammalian cell, using methods wellknown in the art, and recovering the receptor protein after it has beenexpressed in such a host, again using methods well known in the art. Thereceptor may also be isolated from cells which express it, in particularfrom cells which have been transfected with the expression vectorsdescribed below in more detail.

[0043] This invention provides a vector comprising an isolated nucleicacid molecule such as DNA, RNA, or cDNA encoding a human 5-HT_(1F)receptor. Examples of vectors are viruses such as bacteriophages (suchas phage lambda), cosmids, plasmids (such as pUC18, available fromPharmacia, Piscataway, N.J.), and other recombination vectors. Nucleicacid molecules are inserted into vector genomes by methods well known inthe art. For example, insert and vector DNA can both be exposed to arestriction enzyme to create complementary ends on both molecules whichbase pair with each other and are then ligated together with a ligase.Alternatively, linkers can be ligated to the insert DNA which correspondto a restriction site in the vector DNA, which is then digested with therestriction enzyme which cuts at that site. Other means are alsoavailable. A specific example of such plasmids is a plasmid comprisingcDNA having a coding sequence substantially the same as the codingsequence shown in FIG. 1 and designates clone h116a.

[0044] This invention also provides vectors comprising a DNA moleculeencoding a human 5-HT_(1F) receptor, adapted or expression in abacterial cell, a yeast cell, or a mammalian cell which additionallycomprise the regulators elements necessary for expression of the DNA inthe bacterial, yeast, or mammalian cells so located relative to the DNAencoding a human 5-HT_(1F) receptor as to permit expression thereof. DNAhaving coding sequences substantially the same as the coding sequenceshown in FIG. 1 may usefully be inserted into the vectors to expresshuman 5-HT_(1F) receptors. Regulatory elements required for expressioninclude promoter sequences to bind RNA polymerase amino transcriptioninitiation sequences for ribosome binding. For example, a bacterialexpression vector includes a promoter such as the lac promoter and fortranscription initiation the Shine-Dalgarno sequence and the start codonAUG (Maniatis, et al., Molecular Cloning, Cold Spring Harbor Laboratory,1982). Similarly, a eukaryotic expression vector includes a heterologousor homologous promoter or RNA polymerase II, a downstreampolyadenylation signal, the start codon AUG, and a termination codon fordetachment of the ribosome. Such vectors may be obtained commercially orassembled from the sequences described by methods well known in the art,for example the methods described above for constructing vectors ingeneral. Expression vectors are useful to produce cells that express thereceptor. Certain uses for such cells are described in more detailbelow.

[0045] This invention further provides a plasmid adapted for expressionin a bacterial, yeast, or, in particular, a mammalian cell whichcomprises a DNA molecule encoding a human 5-HT_(1F) receptor and theregulatory elements necessary for expression of the DNA in thebacterial, yeast, or mammalian cell so located relative to the DNAencoding a human 5-HT_(1F) receptor as to permit expression thereof.Some plasmids adapted for expression in a mammalian cell are pSVL(available from Pharmacia, Piscataway, N.J.), pcEXV-3 (Miller J. andGermain R. N., J. Exp. Men. 164:1478 (1986)) and pMO5 (Branchek, T. etal, Mol. Pharm. 38:604-609 (1990)). A specific example of such plasmidis a plasmid adapted for expression in a mammalian cell comprising cDNAhaving coding sequences substantially the same as the coding sequenceshown in FIG. 1 and the regulatory elements necessary for expression ofthe DNA in the mammalian cell which is designated pMO5-h116a anddeposited under ATCC Accession No. 75175. Those skilled in the art willreadily appreciate that numerous plasmids adapted for expression in amammalian cell, which comprise DNA of encoding human 5-HT_(1F) receptorsand the regulatory elements necessary to express such DNA in themammalian cell may be constructed utilizing existing plasmids andadapted as appropriate to contain the regulatory elements necessary toexpress the DNA in the mammalian cell. The plasmids may be constructedby the methods described above for expression vectors and vectors ingeneral, and by other methods well known in the art.

[0046] The deposit discussed supra, and the other deposits discussedherein, were made pursuant to, and in satisfaction of, the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedure with the American Type CultureCollection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852.

[0047] This invention provides a mammalian cell comprising a DNAmolecule encoding a human 5-HT_(1F) receptor, such as a mammalian cellcomprising a plasmid adapted for expression in a mammalian cell, whichcomprises a DNA molecule encoding a human 5-HT_(1F) receptor, theprotein encoded thereby is expressed on the cell surface, and theregulatory elements necessary for expression of the DNA in the mammaliancell so located relative to the DNA encoding a human 5-HT_(1F) receptoras to permit expression thereof. Numerous mammalian cells may be used ashosts, including, for example, the mouse fibroblast cell NIH3T3, CHOcells, HeLa cells, Ltk⁻ cells, Y1 cells, etc. A particular example of anLtk⁻ cell is a cell designated L-5-HT_(1F) and deposited under ATCCAccession No. CRL 10957 and comprises the plasmid designated pMO5-h116a.Another example is the murine fibroblast cell line designatedN-5-HT_(1F) and deposited under ATCC Accession No. CRL 10956. Expressionplasmids such as that described supra may be used to transfect mammaliancells by methods well known in the art such as calcium phosphateprecipitation, or DNA encoding these 5-HT_(1F) receptors may beotherwise introduced into mammalian cells, e.g., by microinjection, toobtain mammalian cells which comprise DNA, e.g., cDNA or a plasmid,encoding either human 5-HT_(1F) receptor

[0048] This invention provides a method for determining whether a ligandnot known to be capable of binding to a human 5-HT_(1F) receptor canbind to a human 5-HT_(1F) receptor which comprises contacting amammalian cell comprising a DNA molecule encoding a human 5-HT_(1F)receptor, the protein encoded thereby is expressed on the cell surface,with the ligand under conditions permitting binding of ligands known tobind to the 5-HT_(1F) receptor, detecting the presence of any of theligand bound to the 5-HT_(1F) receptor, and thereby determining whetherthe ligand binds to the 5-HT_(1F) receptor. This invention also providesa method for determining whether a ligand not known to be capable ofbinding to the human 5-HT_(1F) receptor can functionally activate itsactivity or prevent the action of a ligand which does so. This comprisescontacting a mammalian cell comprising an isolated DNA molecule whichencodes a human 5-HT_(1F) receptor with the ligand under conditionspermitting the activator or blockade of a functional response, detectedby means of a bioassay from the mammalian cell such as a secondmessenger response, and thereby detecting whether the ligand activatesor prevents the activation of the human 5-HT_(1F) receptor functionaloutput. The DNA in the cell nay have a coding sequence substantially thesame as the coding sequence shown in FIG. 1 preferably, the mammaliancell is nonneuronal in origin. An example of a nonneuronal mammaliancell is an Ltk⁻ cell, in particular the Ltk⁻ cell designatedL-5-HT_(1F). Another example of a non-neuronal mammalian cell to be usedfor functional assays is a murine fibroblast cell line, specifically theNIH3T3 cell designated N-5-HT_(1F). The preferred method for determiningwhether a ligand is capable of binding to the human 5-HT_(1F) receptorcomprises contacting a transfected nonneuronal mammalian cell (i.e. acell that does not naturally express any type of 5-HT or G-proteincoupled receptor, thus will only express such a receptor if it istransfected into the cell) expressing a 5-HT_(1F) receptor on itssurface, or contacting a membrane preparation derived from such atransfected cell, with the ligand under conditions which are known toprevail, and thus to be associated with, in vivo binding of the ligandsto a 5-HT_(1F) receptor, detecting the presence of any of the ligandbeing tested bound to the 5-HT_(1F) receptor on the surface of the cell,and thereby determining whether the ligand binds to, activates orprevents the activation of the 5-HT_(1F) receptor. This response systemis obtained by transfection of isolated DNA into a suitable host cellcontaining the desired second messenger system such as phosphoinositidehydrolysis, adenylate cyclase, guanylate cyclase or ion channels. Such ahost system is isolated from, preexisting cell lines, or can begenerated by inserting appropriate components of second messengersystems into existing cell lines. Such a transfection system provides acomplete response system for investigation or assay c the activity ofhuman 5-HT_(1F) receptors with ligands as described above. Transfectionsystems are useful as living cell cultures for competitive bindingassays between known or candidate drugs and ligands which bind to thereceptor and which are labeled by radioactive, spectroscopic or otherreagents. Membrane preparations containing the receptor isolated fromtransfected cells are also useful for these competitive binding assays.Functional assays of second messenger systems or their sequelae intransfection systems act as assays for binding affinity and efficacy inthe activation of receptor function. A transfection system constitutes a“drug discovery system” useful for the identification of natural orsynthetic compounds with potential for drug development that can befurther modified or used directly as therapeutic compounds to activateor inhibit the natural functions of the human 5-HT_(1F) receptor. Thetransfection system is also useful for determining the affinity andefficacy of known drugs at the human 5-HT_(1F) receptor sites.

[0049] This invention also provides a method of screening drugs toidentify drugs which specifically interact with, and bind to, the human5-HT_(1F) receptor on the surface of a cell which comprises contacting amammalian cell comprising a DNA molecule encoding a human 5-HT_(1F)receptor on the surface of a cell with a plurality of drugs, determiningthose drugs which bind to the mammalian cell, and thereby identifyingdrugs which specifically interact with, and bind to, the human 5-HT_(1F)receptor. This invention also provides a method of screening drugs toidentify drugs which interact with, and activate or block the activationof, the human 5-HT_(1F) receptor on the surface of a cell whichcomprises contacting the mammalian cell comprising an isolated DNAmolecule encoding and expressing a human 5-HT_(1F) receptor with aplurality of drugs, determining those drugs which activate or block theactivation of the receptor in the mammalian cell using a bioassay suchas a second messenger assays, and thereby identifying drugs whichspecifically interact with, and activate or block the activation of, ahuman 5-HT_(1F) receptor. The DNA in the cell may have a coding sequencesubstantially the same as the coding sequence shown in FIG. 1 (Seq. I.D.No. 1). Preferably, the mammalian cell is nonneuronal in origin. Anexample of a nonneuronal mammalian cell is an Ltk⁻ cell, in particularthe Ltk⁻ cell designated L-5-HT_(1F). Another example of a non-neuronalmammalian cell to be used for functional assays is a murine fibroblastcell line, specifically the NIH3T3 cell designated N-5-HT_(1F). Drugcandidates are identified by choosing chemical compounds which bind withhigh affinity to the expressed 5-HT_(1F) receptor protein in transfectedcells, using radioligand binding methods well known in the art, examplesof which are shown in the binding assays described herein. Drugcandidates are also screened for selectivity by identifying compoundswhich bind with high affinity to one particular 5-HT_(1F) receptorsubtype but do not bind with high affinity to any other serotoninreceptor subtype or to any other known receptor site. Because selective,high affinity compounds interact primarily with the target 5-HT_(1F)receptor site after administration to the patient, the chances ofproducing a drug with unwanted side effects are minimized by thisapproach. This invention provides a pharmaceutical compositioncomprising a drug identified by the method described above and apharmaceutically acceptable carrier. As used herein, the term“pharmaceutically acceptable carrier” encompasses any of the standardpharmaceutical carriers, such as a phosphate buffered saline solution,water, and emulsions, such as an oil/water or water/oil emulsion, andvarious types of wetting agents. Once the candidate drug has been shownto be adequately bio-available following a particular route ofadministration, for example orally or by injection (adequate therapeuticconcentrations must be maintained at the site of action for an adequateperiod to gain the desired therapeutic benefit), and has been shown tobe non-toxic and therapeutically effective in appropriate diseasemodels, the drug may be administered to patients by that route ofadministration determined to make the drug bio-available, in anappropriate so id or solution formulation, to gain the desiredtherapeutic benefit.

[0050] This invention provides a nucleic acid probe comprising a nucleicacid molecule of at least 15 nucleotides capable of specificallyhybridizing with a sequence included within the sequence of a nucleicacid molecule encoding a human 5-HT_(1F) receptor, for example with acoding sequence included within the sequence shown in FIG. 1. As usedherein, the phrase “specifically hybridizing” means the ability of anucleic acid molecule to recognize a nucleic acid sequence complementaryto its own and to form double-helical segments through hydrogen bondingbetween complementary base pairs. Nuclei acid probe technology is wellknown to those skilled in the art who will readily appreciate that suchprobes may vary greatly in length and may be labeled with a detectablelabel, such as a radioisotope or fluorescent dye, to facilitatedetection of the probe. Detection of nucleic acid encoding human5-HT_(1F) receptors is useful as a diagnostic test for any diseaseprocess in which levels of expression of the corresponding 5-HT_(1F)receptor is altered. DNA probe molecules are produced by insertion of aDNA molecule which encodes human 5-HT_(1F) receptor or fragments thereofinto suitable vectors, such as plasmids or bacteriophages, followed byinsertion into suitable bacterial host cells and replication andharvesting of the DNA probes, all using methods well known in the art.For example, the DNA may be extracted from a cell lysate using phenoland ethanol, digested with restriction enzymes corresponding to theinsertion sites of the DNA into the vector (discussed above),electrophoresed, and cut out of the resulting gel. An example of suchDNA molecule is shown in FIG. 1. The probes are useful for ‘in situ’hybridization or in order to locate tissues which express this genefamily, or for other-hybridization assays for the presence of thesegenes or their mRNA in various biological tissues. In addition,synthesized oligonucleotides (produced by a DNA synthesizer)complementary to the sequence of a DNA molecule which encodes human5-HT_(1F) receptor of are useful as probes for these genes, for theirassociated mRNA, or for the isolation of related genes by homologyscreening of genomic or cDNA libraries, or by the use of amplificationtechniques such as the Polymerase chain Reaction. Synthesizedoligonucleotides as described may also be used to determine the cellularlocalization of the mRNA produced by the 5-HT_(1F) gene by in situhybridization. An example of such an oligonucleotide is:5′-TCTCACCACTCTCCAAAACAGGACTTCACCTCCTCCTG-3′ (Seq. I.D. No. 9).

[0051] This invention also provides a method of detecting expression ofa 5-HT_(1F) receptor on the surface of a cell by detecting the presenceof mRNA coding for a 5-HT_(1F) receptor which comprises obtaining totalmRNA from the cell using methods well known in the art and contactingthe mRNA so obtained with a nucleic acid probe comprising a nucleic acidmolecule of at least 15 nucleotides capable of specifically hybridizingwith a sequence included within the sequence of a nucleic acid moleculeencoding a human 5-HT_(1F) receptor under hybridizing conditions,detecting the presence of mRNA hybridized to the probe, and therebydetecting the expression of the 5-HT_(1F) receptor by the cell.Hybridization of probes to target nucleic acid molecules such as mRNAmolecules employs techniques well known in the art. In one possiblemeans of performing this method, nucleic acids are extracted byprecipitation from lysed cells and the mRNA is isolated from the extractusing a column which binds the poly-A tails of the mRNA molecules. ThemRNA is then exposed to radioactively labelled probe on a nitrocellulosemembranes and the probe hybridizes to and thereby labels complementarymRNA sequences. Binding may be detected by autoradiography orscintillation counting. However, other methods for performing thesesteps are well known to those skilled in the art, and the discussionabove is merely an example.

[0052] This invention provides an antisense oligonucleotide having asequence capable of binding specifically with any sequences of an nRNAmolecule which encodes a human 5-HT_(1F) receptor so as to preventtranslation of the mRNA molecule. The antisense oligonucleotide may havea sequence capable of binding specifically with any sequences of thecDNA molecule whose sequence is shown in FIG. 1. As used herein, thephrase “binding specifically” means the ability of a nucleic acidsequence to recognize a nucleic acid sequence complementary to its ownand to form double-helical segments through hydrogen bonding betweencomplementary base pairs. A particular example of an antisenseoligonucleotide is an antisense oligonucleotide comprising chemicalanalogues of nucleotides.

[0053] This invention also provides a pharmaceutical compositioncomprising an amount of the oligonucleotide described above effective toreduce expression of a human 5-HT_(1F) receptor by, passing through acell membrane and binding specifically with mRNA encoding a human5-HT_(1F) receptor in the cell so as to prevent its translation and apharmaceutically acceptable hydrophobic carrier capable of passingthrough a cell membrane. The oligonucleotide may be coupled to asubstance which inactivates mRNA, such as a ribozyme. Thepharmaceutically acceptable hydrophobic carrier capable of passingthrough cell membranes may also comprise a structure which binds to areceptor specific for a selected cell type and is thereby taken up bycells of the selected cell type. The structure may be part of a proteinknown to bind a cell-type specific receptor, for example an insulinmolecule, which would target pancreatic cells. DNA molecules havingcoding sequences substantially the same as the coding sequence shown inFIG. 1 may be used as the oligonucleotides of the pharmaceuticalcomposition.

[0054] This invention also provides a method of treating abnormalitieswhich are alleviated by reduction of expression of a 5-HT_(1F) receptorwhich comprises administering to a subject an amount of thepharmaceutical composition described above effective to reduceexpression of the 5-HT_(1F) receptor by the subject. This inventionfurther provides a method of treating an abnormal condition related to5-HT_(1F) receptor activity which comprises administering to a subjectan amount of the pharmaceutical composition described above effective toreduce expression of the 5-HT_(1F) receptor by the subject. Severalexamples of such abnormal conditions are dementia, Parkinson's disease,feeding disorders, pathological anxiety, schizophrenia, or a migraineheadache.

[0055] Antisense oligonucleotide drugs inhibit translation of mRNAencoding these receptors. Synthetic oligonucleotides, or other antisensechemical structures are designed to bind to mRNA encoding the 5-HT_(1F)receptor and inhibit translation of mRNA and are useful as drugs toinhibit expression of 5-HT_(1F) receptor genes in patients. Thisinvention provides a means to therapeutically alter levels of expressionof human 5-HT_(1F) receptors by the use of a synthetic ant-,senseoligonucleotide drug (SAOD) which inhibits translation of mRNA encodingthese receptors. Synthetic oligonucleotides, or other antisense chemicalstructures designed to recognize and selectively bind to mRNA, areconstructed to be complementary to portions If the nucleotide sequencesshown in FIG. 1 of DNA, RNA or of chemically modified, artificialnucleic acids. The SAOD is designed to be stable in the blood stream foradministration to patients by injection, or in laboratory cell cultureconditions, for administration to cells removed from the patient. TheSAOD is designed to be capable of passing through cell membranes inorder to enter the cytoplasm of the cell by virtue of physical andchemical properties of the SAOD which render it capable of passingthrough cell membranes (e.g. by designing small, hydrophobic SAODchemical structures) or by virtue of specific transport systems in thecell which recognize and transport the SAOD into the cell. In addition,the SAOD can be designed for administration only to certain selectedcell populations by targeting the SAOD to be recognized by specificcellular uptake mechanisms which binds and takes up the SAOD only withincertain selected cell populations. For example, the SAOD may be designedto bind to a receptor found only in a certain cell type, as discussedabove. The SAOD is also designed to recognize and selectively bind tothe target mRNA sequence, which may correspond to a sequence containedwithin the sequence shown in FIG. 1 by virtue of complementary basepairing to the mRNA. Finally, the SAOD is designed to inactivate thetarget mRNA sequence by any of three mechanisms: 1) by binding to thetarget mRNA and thus inducing degradation of the mRNA by intrinsiccellular mechanisms such as RNAse I digestion, 2) by inhibitingtranslation of the mRNA target by interfering with the binding oftranslation-regulating factors or of ribosomes, or 3) by inclusion ofother chemical structures, such as ribozyme sequences or reactivechemical groups, which either degrade or chemically modify the targetmRNA. Synthetic antisense oligonucleotide drugs have been shown to becapable of the properties described above when directed against CANtargets (J. S. Cohen, Trends in Pharm. Sci. 10, 435 (1989); H. M.Weintraub, Sci. Am. January (1990) p. 40). In addition, coupling ofribozymes to antisense oligonucleotides is a promising strategy forinactivating target mRNA (N. Sarver et al., Science 247, 1222 (1990)).An SAOD serves as an effective therapeutic agent if it is designed to beadministered to a patient by injection, or if the patient's target cellsare removed, treated with the SAOD in the laboratory, and replaced inthe patient. In this manner, an SAOD serves as a therapy to reducereceptor expression in particular target cells of a patient, in anyclinical condition which may benefit from reduced expression of5-HT_(1F) receptors.

[0056] This invention provides an antibody directed to the human5-HT_(1F) receptor, for example a monoclonal antibody directed to anepitope of a human 5-HT_(1F) receptor present on the surface of a celland having an amino acid sequence substantially the same as an aminoacid sequence for a cell surface epitope of the human 5-HT_(1F) receptorincluded in the amino acid sequence shown in FIG. 1 (Seq. I.D. Nos. 2,7). Amino acid sequences may be analyzed by methods well known in theart to determine whether they produce hydrophobic or hydrophilic regionsin the proteins which they build. In the case of cell membrane proteins,hydrophobic regions are well known to form the part of the protein thatis inserted into the lipid bilayer which forms the cell membrane, whilehydrophilic regions are located on the cell surface, in an aqueousenvironment. Therefore antibodies to the hydrophilic amino acidsequences shown in FIG. 1 will bind to a surface epitope of a human5-HT_(1F) receptor, as described. Antibodies directed to human 5-HT_(1F)receptors may be serum-derived or monoclonal and are prepared usingmethods well known in the art. For example, monoclonal antibodies areprepared using hybridoma technology by fusing antibody producing B cellsfrom immunized animals with myeloma cells and selecting the resultinghybridoma cell line producing the desired antibody. Cells such as NIH3T3cells or Ltk⁻ cells may be used as immunogens to raise such an antibody.Alternatively, synthetic peptides may be prepared using commerciallyavailable machines and the amino acid sequence shown in FIG. 1. As astill further alternative, DNA, such as a cDNA or a fragment thereof,may be cloned and expressed and the resulting polypeptide recovered andused as an immunogens. These antibodies are useful to detect thepresence of human 5-HT_(1F) receptors encoded by the isolated DNA, or toinhibit the function of the receptors in living animals, in humans, orin biological tissues or fluids isolated from animals or humans.

[0057] This invention provides a pharmaceutical composition whichcomprises an amount of an antibody directed to the human 5-HT_(1F)receptor effective to block binding of naturally occurring ligands tothe 5-HT_(1F) receptor, and a pharmaceutically acceptable carrier. Amonoclonal antibody directed to an epitope of a human 5-HT_(1F) receptorpresent on the surface of a cell and having an amino acid sequencesubstantially the same as an amino acid sequence for a cell surfaceepitope of the human 5-HT_(1F) receptor included in the amino acidsequence shown in FIG. 1 is useful for this purpose.

[0058] This invention also provides a method of treating abnormalitieswhich a alleviated by reduction of expression of a human 5-HT_(1F)receptor which comprises administering to a subject an amount of thepharmaceutical composition described above effective to block binding ofnaturally occurring ligands to the 5-HT_(1F) receptor and therebyalleviate abnormalities resulting from overexpression of a human5-HT_(1F) receptor. Binding of the antibody to the receptor prevents thereceptor from functioning, thereby neutralizing the effects ofoverexpression. The monoclonal antibodies describe. above are bothuseful for this purpose. This invention additionally provides a methodof treating an abnormal condition related to an excess of 5-HT_(1F)receptor activity which comprises administering to a subject an amountof the pharmaceutical composition described above effective to blockbinding of naturally occurring ligands to the 5-HT_(1F) receptor andthereby alleviate the abnormal condition. Some examples of abnormalconditions are dementia, Parkinson's disease, feeding disorders,pathological anxiety, schizophrenia, and a migraine headache.

[0059] This invention provides a method of detecting the presence of a5-HT_(1F) receptor on the surface of a cell which comprises contactingthe cell with an antibody directed to the human 5-HT_(1F) receptor,under conditions permitting binding of the antibody to the receptor,detecting the presence of the antibody bound to the cell, and therebythe presence of the human 5-HT_(1F) receptor on the surface of the cell.Such a method is useful for determining whether a given cell isdefective in expression of 5-HT_(1F) receptors on the surface of thecell. Bound antibodies are detected by methods well known in the art,for example by binding fluorescent markers to the antibodies andexamining the cell sample under a fluorescence microscope to detectfluorescence on a cell indicative of antibody binding. The monoclonalantibodies described above are useful for this purpose.

[0060] This invention provides a transgenic nonhuman mammal expressingDNA encoding a human 5-HT_(1F) receptor. This invention also provides atransgenic nonhuman mammal expressing DNA encoding a human 5-HT_(1F)receptor so mutated as to be incapable of normal receptor activity, andnot expressing native 5-HT_(1F) receptor. This invention also provides atransgenic nonhuman mammal whose genome comprises antisense DNAcomplementary to DNA encoding a human 5-HT_(1F) receptor so placed as tobe transcribed into antisense mRNA which is complementary to R Aencoding a 5-HT_(1F) receptor and which hybridizes to mRNA encoding a5-HT_(1F) receptor thereby reducing its translation. The DNA mayadditionally comprise an inducible promoter or additionally comprisetissue specific regulators elements, so that expression can be induced,or restricted to specific cell types. Examples of DNA are DNA or cDNAmolecules having a coding sequence substantially the same as the codingsequence shown in FIG. 1 (Seq. I.D. No. 1). An example of a transgenicanimal is a transgenic mouse. Examples of tissue specificity-determiningregions are the metallothionein promotor (Low, M. J., Lechan, R. M.,Hammer, R. E. et al. Science 231:1002-1004 (1986)) and the L7 promotor(Oberdick, J., Smeyne, R. Z., Mann, J. R., Jackson, S. and Morgan, J. I.Science 248:223-226 (1990)).

[0061] Animal model systems which elucidate the physiological andbehavioral roles of human 5-HT_(1F) receptors are produced by creatingtransgenic animals in which the expression of a 5-HT_(1F) receptor iseither increased or decreased, or the amino aid sequence of theexpressed 5-HT_(1F) receptor protein is altered, by a variety oftechniques. Examples of these techniques include: 1) Insertion of normalor mutant versions of DNA encoding a human 5-HT_(1F) receptor orhomologous animal versions of these genes, by microinjection, retroviralinfection or other means well known to those skilled in the art, intoappropriate fertilized embryos in order to provide a transgenic animal(Hogan B. et al. Manipulating the Mouse Embryo, A Laboratory Manual,Cold Spring Harbor Laboratory (1986)). 2) Homologous recombination(Capecchi M. R. Science 244:1288-1292 (1989); Zimer, A. and Gruss, P.Nature 338:150-153 (1989)) of mutant or normal, human or animal versionsof these genes with the native gene locus in transgenic animals to alterfine regulation of expression or the structure of these 5-HT_(1F)receptors. The technique of homologous recombination is well known inthe art. It replaces the native gene with the inserted gene and so isuseful for producing an animal that cannot express native receptor butdoes express, for example, an inserted mutant receptor, which hasreplaced the native receptor in the animal's genome by recombination,resulting in underexpression of the receptor. Microinjection adds genesto the genome, but does not remove them, and so is useful for producingan animal which expresses its own and added receptors, resulting inoverexpression of the receptor. One means available for producing atransgenic animal, with a mouse as an example, is as follows: Femalemice are mated, and the resulting fertilized eggs are dissected out oftheir oviducts. The eggs are stored in an appropriate medium such as M2medium (Hogan B. et al. Manipulating the Mouse Embryo, A LaboratoryManual, Cold Spring Harbor Laboratory (1986)). DNA or cDNA encoding ahuman 5-HT_(1F) receptor is purified from a vector (such as plasmidpMO5-h116a described above) by methods well known in the art. Induciblepromoters may be fused with the coding region of the DNA to provide anexperimental means to regulate expression of the trans-gene.Alternatively or in addition, tissue specific regulatory elements may befused with the coding region to permit tissue-specific expression of thetrans-gene. The DNA, in an appropriately buffered solution, is put intoa microinjection needle (which may be made from capillary tubing using apipet puller) and the egg to be injected is put in a depression slide.The needle is Inserted into the pronucleus of the egg, and the DNAsolution is injected. The infected egg is then transferred into theoviduct of a pseudopregnant mouse (a mouse stimulated by the appropriatehormones to maintain pregnancy but which is not actually pregnant),where it proceeds to the uterus, implants, and develops to term. Asnoted above, microinjection is not the only method for inserting DNAinto the egg cell, and is used here only for exemplary purposes.

[0062] Since the normal action of receptor-specific drugs is to activateor to inhibit the receptor, the transgenic animal model systemsdescribed above are useful for testing the biological activity of drugsdirected against these 5-HT_(1F) receptors even before such drugs becomeavailable. These animal model systems are useful for predicting orevaluating possible therapeutic applications of drugs which activate orinhibit these 5-HT_(1F) receptors by inducing or inhibiting expressionof the native or trans-gene and thus increasing or decreasing expressionof normal or mutant 5-HT_(1F) receptors in the living animal. Thus, amodel system is produced in which the biological activity of drugsdirected against these 5-HT_(1F) receptors are evaluated before suchdrugs become available. The transgenic animals which over or underproduce the 5-HT_(1F) receptor indicate by their physiological statewhether over or under production of the 5-HT_(1F) receptor istherapeutically useful. It is therefore useful to evaluate drug actionbased on the transgenic model system. :One use is based on the fact thatit is well known in the art that a drug such as an antidepressant actsby blocking neurotransmitter uptake, and thereby increases the amount ofneurotransmitter in the synaptic cleft. The physiological result of thisaction is to stimulate the production of less receptor by the affectedcells, leading eventually to underexpression. Therefore, an animal whichunderexpresses receptor is useful as a test system to investigatewhether the actions of such drugs which result in under expression arein fact therapeutic, Another use is that if overexpression is found tolead to abnormalities, then a drug which down-regulates or acts as anantagonist to 5-HT_(1F) receptor is indicated as worth developing, andif a promising therapeutic application is uncovered by these animalmodel systems, activation or inhibition of the 5-HT_(1F) receptor isachieved therapeutically either by producing agonist or antagonist drugsdirected against these 5-HT_(1F) receptors or by any method whichincreases or decreases the expression of these 5-HT_(1F) receptors inman.

[0063] This invention provides a method of determining the physiologicaleffects of expressing varying levels of human 5-HT_(1F) receptors whichcomprises producing a transgenic nonhuman animal whose levels of human5-HT_(1F) receptor expression are varied by use of an inducible promoterwhich regulates human 5-HT_(1F) receptor expression. This invention alsoprovides a method of determining the physiological effects of expressingvarying levels of human 5-HT_(1F) receptors which comprises producing apanel of transgenic nonhuman animals each expressing a different amountof human 5-HT_(1F) receptor. Such animals may be produced by introducingdifferent amounts of DNA encoding a human 5-HT_(1F) receptor into theoocytes from which the transgenic animals are developed.

[0064] This invention also provides a method for identifying a substancecapable of alleviating abnormalities resulting from overexpression of ahuman 5-HT_(1F) receptor comprising administering the substance to atransgenic nonhuman mammal expressing a least one artificiallyintroduced DNA molecule encoding a human 5-HT_(1F) receptor anddetermining whether the substance alleviates the physical and behavioralabnormalities displayed by the transgenic nonhuman mammal as a result ofoverexpression of a human 5-HT_(1F) receptor. As used herein, the term“substance” means a compound or composition which may be natural,synthetic, or a product derived from screening. Examples of DNAmolecules are DNA or cDNA molecules having a coding sequencesubstantially the same as, the coding sequence shown in FIG. 1.

[0065] This invention provides a pharmaceutical composition comprisingan amount of the substance described supra effective to alleviate theabnormalities resulting from overexpression of 5-HT_(1F) receptor and apharmaceutically acceptable carrier.

[0066] This invention further provides a method for treating theabnormalities resulting from overexpression of a human 5-HT_(1F)receptor which comprises administering to a subject an amount of thepharmaceutical composition described above effective to alleviate theabnormalities resulting from overexpression of a human 5-HT_(1F)receptor.

[0067] This invention provides a method for identifying a substancecapable of alleviating the abnormalities resulting from underexpressionof a human 5-HT_(1F) receptor comprising administering the substance tothe transgenic nonhuman mammal described above which expresses onlynonfunctional human 5-HT_(1F) receptor and determining whether thesubstance alleviates the physical and behavioral abnormalities displayedby the transgenic nonhuman mammal as a result of underexpression of ahuman 5-HT_(1F) receptor.

[0068] This invention also provides a pharmaceutical compositioncomprising an amount of a substance effective to alleviate abnormalitiesresulting from underexpression of 5-HT_(1F) receptor and apharmaceutically acceptable carrier.

[0069] This invention further provides a method for treating theabnormalities resulting from, underexpression of a human 5-HT_(1F)receptor which comprises administering to a subject an amount of thepharmaceutical composition described above effective to alleviate theabnormalities resulting from underexpression of a human 5-HT_(1F)receptor.

[0070] This invention provides a method for diagnosing a predispositionto a disorder associated with the expression of a specific human5-HT_(1F) receptor allele which comprises: a) obtaining DNA of subjectssuffering from the disorder; b) performing a restriction digest of theDNA with a panel of restriction enzymes; c.electrophoreticallyseparating the resulting DNA fragments on a sizing gel; d) contactingthe resulting gel with a nucleic acid probe capable of specificallyhybridizing to DNA encoding a human 5-HT_(1F) receptor and labelled witha detectable marker; e) detecting labelled bands which have hybridizedto the DNA encoding a human 5-HT_(1F) receptor labelled with adetectable marker to create a unique band pattern specific to the DNA ofsubjects suffering from the disorder; f) preparing DNA obtained fordiagnosis by steps a-e; and g) comparing the unique band patternspecific to the DNA of subjects suffering from the disorder from step eand the DNA obtained for diagnosis from step f to determine whether thepatterns are the same or different and thereby to diagnosepredisposition to the disorder if the patterns are the same. This methodmay also be used to diagnose a disorder associated with the expressionof a specific human 5-HT_(1F) receptor allele.

[0071] This invention provides a method of preparing the isolated5-HT_(1F) receptor which comprises inducing cells to express 5-HT_(1F)receptor, recovering the receptor from the resulting cells, andpurifying the receptor so recovered. An example of an isolated 5-HT_(1F)receptor as an isolated protein having substantially the same amino acidsequence as the amino acid sequence shown in FIG. 1 (Seq. I.D. Nos. 2,7). For example, cells can be induced to express receptors by exposureto substances such as hormones. The cells can then be homogenized andthe receptor isolated from the homogenate using a affinity columncomprising, for example, serotonin or another substance which is knownto bind to the receptor. The resulting fractions can then be purified bycontacting them with an ion exchange column, and determining whichfraction contains receptor activity or binds anti-receptor antibodies.

[0072] This invention provides a method of preparing the isolated5-HT_(1F) receptor which comprises inserting nucleic acid encoding5-HT_(1F) receptor in a suitable vector, inserting the resulting vectorin a suitable host cell, recovering the receptor produced by theresulting cell, and purifying the receptor so recovered. An example ofan isolated 5-HT_(1F) receptor is an isolated protein havingsubstantially the same amino acid sequence as the amino acid sequenceshown in FIG. 1. This method for preparing 5-HT_(1F) receptor usesrecombinant DNA technology methods well known in the art. For example,isolated nucleic acid encoding 5-HT_(1F) receptor is inserted in asuitable vector, such as an expression vector. A suitable host cell,such as a bacterial cell, or a eukaryotic cell such as a yeast cell, istransfected with the vector. 5-HT_(1F) receptor is isolated from theculture medium by affinity purification or by chromatography or by othermethods well known in the art.

[0073] This invention provides an antisense oligonucleotide having asequence capable of binding specifically with any sequences of an nRNAmolecule which encodes a receptor so as to prevent translation of hemRNA molecule (Seq. I.D. No. 9).

[0074] This invention also provides a transgenic nonhuman mammalexpressing DNA encoding a receptor.

[0075] This invention further provides a transgenic nonhuman mammalexpressing DNA encoding a receptor so mutated as to be incapable ofnormal receptor activity, and not expressing native receptor.

[0076] This invention provides a method of determining the physiologicaleffects of expressing varying levels of a receptor which comprisesproducing a transgenic nonhuman animal whose levels of receptorexpression are varied by use of an inducible promoter which regulatesreceptor expression.

[0077] This invention also provides a method of determining thephysiological effects of expressing varying levels of a receptor whichcomprises producing a panel of transgenic nonhuman animals eachexpressing a different amount of the receptor.

[0078] This invention further provides transgenic nonhuman mammal whosegenome comprises antisense DNA complementary to DNA encoding a receptorso placed as to be transcribed into antisense mRNA which iscomplementary to mRNA encoding the receptor and which hybridizes to mRNAencoding the receptor thereby preventing its translation.

[0079] This invention provides a method for determining whether a ligandnot known to be capable of binding to a receptor can bind to a receptorwhich comprises contacting a mammalian cell comprising an isolated DNAmolecule encoding the receptor with the ligand under conditionspermitting binding of ligands known to bind to a receptor, detecting thepresence of any of the ligand bound to the receptor, and therebydetermining whether the ligand binds to the receptor.

[0080] Applicants have identified individual receptor subtype proteinsand have described methods for the identification of pharmacologicallycompounds for therapeutic treatments. Pharmacological compounds whichare directed against specific receptor subtypes provide effective newtherapies with minimal side effects.

[0081] This invention identifies for the first time a new receptorprotein, its amino acid sequence, and its human gene. Furthermore, thisinvention describes a previously unrecognized group of receptors withinthe definition of a 5-HT_(1F) receptor. The information and experimentaltools provided by this discovery are useful to generate new therapeuticagents, and new therapeutic or diagnostic assays for this new receptorprotein, its associated mRNA molecule or its associated genomic DNA. Theinformation and experimental tools provided by this discovery will beuseful to generate new therapeutic agents, and new therapeutic ordiagnostic assays for this new receptor protein, its associated mRNAmolecule, or its associated genomic DNA.

[0082] Specifically, this invention relates to the first isolation of ahuman cDNA and genomic clone encoding a 5-HT_(1F) receptor. A new humangene for the receptor identified herein as 5-HT_(1F) has been identifiedand characterized, and a series of related cDNA and genomic clones havebeen isolated. In addition, the human 5-HT_(1F) receptor has beenexpressed in Ltk⁻ cells and NIH3T3 cells by transfecting the cells withthe plasmid pMO5-h116a. The pharmacological binding properties of theprotein encoded have been determined, and these binding propertiesclassify this protein as a serotonin 5-HT_(1F) receptor. Mammalian celllines expressing this human 5-HT_(1F) receptor at the cell surface havebeen constructed, thus establishing the first well-defined, culturedcell lines with which to study this 5-HT_(1F) receptor.

[0083] The invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more full in the claims whichfollow thereafter.

EXPERIMENTAL DETAILS

[0084] Materials and Methods

[0085] Polymerase Chain Reaction (PCR): The third (III, and fifth (V)transmembrane domains of the following receptors were aligned and usedto synthesize a pair of “degenerate” primers: 5-HT_(1A) (Seq. I.D. No.3), 5-HT_(1C) (Seq. I.D. No. 4), 5-HT₂ (Seq. I.D. No. 8) and the5-HT_(1Dα/β) (Seq. I.D. Nos. 5 and 6, respectively) receptors (patentpending). These primers hybridize to opposite strands of targetsequences to allow amplification of the region between the correspondingtransmembrane domains. That primer which was designed to anneal totransmembrane domain III is designated 3.17 and consists of a mixture of192 different 31-mers with two inosine nucleotides; the primer whichannealed to transmembrane domain V is designated 5.5 and consists of amixture of 288 different 27-mers with five inosine nucleotides. EcoRIlinkers were included at the 5′ end of primer 3.1, to facilitate thesubcloning of the amplified cDNA in pBluescript (Stratagene) vectors. 5μg of poly (A+) RNA from rat brain was reverse transcribed by avianmyeloblastosis virus reverse transcriptase (AMV) including 3 μM each of3.17 and 5.5 primers. The resulting single-stranded cDNA was used in aPCR reaction under the following conditions: 94° C. for 1 minute, 50° C.for 2 minutes and 72° C. for 3 minutes for 40 cycles. Following PCR, 90μl of the reaction was phenolichloroform extracted and precipitated; 10μl was visualized on a gel using ethidium bromide staining. Afterprecipitation the sample was treated with T4 DNA polymerase and digestedwith EcoR1 prior to separation on a 1% agarose gel. The DNA fragment wasisolated from the gel, kinased and cloned into pBluescript. Recombinantclones were analyzed by sequencing.

[0086] Cloning and Sequencing: A human lymphocyte genomic library(Stratagene) was screened using the rat S51 fragment (obtained by PCR)as a probe. The probe was labeled with ³²P by the method of randompriming (Feinberg et al., 1983). Hybridization was performed a 50° C. ina solution containing 50% formamide, 10% dexran sulfate, 5×S5C (1×SSC is0.5 M sodium, chloride, 0.015 M sod50A. citrate), 1×Denhart's (0.02%polyvinylpyrrolidone, 0.02% Ficoll, and 0.02% bovine serum albumin), and200 μg/ml of sonicated salmon sperm DNA. The filters were washed at 50°C. in 0.1×SSC containing 0.1% sodium doaecyl sulfate (SDS) and exposedat −70° C. to Kodak XAR film in the presence of an intensifying screen.Lambda phage hybridizing to the probe were plaque purified and DNA wasprepared for Southern blot analysis (Southern, 1975; Maniatis et al.,1982). For subcloning and further Southern blot analysis DNA wasinserted Into pUC18 (Pharmacia, Piscataway, N.J.). Nucleotide sequenceanalysis was done by the Sanger dideoxy nucleotide chain-terminationmethod (Sancer 1977) or, denatured double-stranded plasmid templatesusing Sequenase (U.S. Biochemical Corp., Cleveland, Ohio).

[0087] Expression: The entire coding region of clone h116a was clonedinto the eukaryotic expression vector pcEXV-3 (Miller, 1986). Stablecell lines were obtained by cotransfection with the plasmid pcEXV-3(containing the 5-HT_(1F) receptor gene) and the plasmid pGCcos3neo(containing the aninoglycoside transferase gene) into Ltk⁻ cells orNIH3T3 cells using calcium phosphate (reagents obtained from SpecialtyMedia, Lavellette, N.J.) The cells were grow in a controlled environment(37° C., 5% CO₂) as monolayers in Dulbecco's modified Eagle medium(Gibco, Grand Island, N.Y.) containing 25 mM glucose and supplementedwith 10% bovine calf serum, 100 U/ml penicillin G and 100 μg/mlstreptomycin sulfate. Stable clones were then selected for resistance tothe antibiotic G-418 and harvested membranes were screened for theirability to bind [³H] serotonin.

[0088] Membrane Preparation: Membranes were prepare from transfectedLtk⁻ cells which were grown to 100% confluency. The cells were washedtwice with phosphate-buffered saline, scraped from the culture dishes oml of ice-cold phosphate-buffered saline, and centrifuged at 200×g for 5min at 4°. The pellet was resuspended in 2.5 ml of ice-cold Tris buffer(20 mM. Tris-HCl, pH 7.4 at 23°, 5 mM EDTA) and homogenized by a Wheatontissue grinder. The lysate was subsequently centrifuged at 200×g for 5min at 4° C. to pellet large fragments which were discarded. Thesupernatant was collected and centrifuged at 40,000×g for 20 min at 4°.The pellet resulting from this centrifugation was washed once inice-cold Tris wash buffer and finally resuspended in a final buffercontaining 50 ml Tris-HCl and 0.5 my EDTA, pH 7.4 at 23°. Membranepreparations were kept on ice and utilized within two hours for theradioligand binding assays. Protein concentrations were determined bythe method of Bradford (1976) using bovine serum albumin as thestandard.

[0089] Radioligand Binding: [³H]5HT binding was performed using slightmodifications of the 5-HT_(1D) assay conditions reported byHerrick-Davis and Titeler (1988) with the omission of masking ligands.Radioligand binding studies were achieved at 3° C. in a total volume of250 μl of buffer (50 mM Tris, 10 mM MgCl₂, 0.2 mM EDTA, 10 μM pargyline,0.1 % ascorbate, pH 7.4 at 37° C.) in 96 well microtiter plates.Saturation studies were conducted using [³H]5-HT at 12 differentconcentrations ranging from 0.5 nM to 100 nM. Displacement studies wereperformed using 4.5-5.5 nM [³H]5-HT. The binding profile of drugs incompetition experiments was accomplished using 10-12 concentrations ofcompound. Incubation times were 30 min for both saturation anddisplacement studies base, upon initial investigations which determinedequilibrium binding conditions. Nonspecific binding was defined in thepresence of 10 μM 5-HT. Binding was initiated by the addition of 50 μlmembrane homogenates (10-20 μg). The reaction was terminated by rapidfiltration through presoaked (0.5% polyethyleneimine filters using 48RCell Brandel Harvester (Gaithersburg Md.). Subsequently, filters werewashed for 5 sec with ice cold buffer (50 mM Tris HCL, pH 7.4 at 4° C.),dried and placed into vials containing 2.5 ml of Read-Safe (Beckman,Fullerton, Calif.) and radioactivity was measured using a Beckman LS5000A liquid scintillation counter. The efficiency of counting of[³H]5HT averaged between 45-50%. Binding data was analyzed bycomputer-assisted nonlinear regression analysis (Accufit and Accucomp,Lundon Software, Chagrin Falls, Ohio). IC₅₀ values were converted to K₁values using the Cheng-Prusoff equation (1973). All experiments wereperformed in triplicate.

[0090] Measurement of cAMP Formation

[0091] Transfected NIH3T3 cells (estimated Bmax from one pointcompetition studies=448 fmol/g of protein, were incubated in DMEM, 5 nMtheophyline, 10 mM Hepes [4-(2-Hydroxyethyl]-1-piperazineethanesulfonicacid), 10 μM pargyline, for 20 minutes at 37° C., 5% CO2. Drugdose-effect curves were then conducted by adding 6 different finalconcentrations of drug, followed immediately by the addition offorskolin (10 μM). Subsequently, the cells were incubated for anadditional 10 minutes at 37° C., 5% CO2. The media was aspirated and thereaction terminated by the addition of 100 mM HCl. The plates werestored at 4° C. for 15 minutes and centrifuged for 5 minutes (500×g at4° C.) to pellet cellular debris. Aliquots of the supernatant fractionwere then stored at −20° C. prior to assessment of cAMP formation byradioimmunoassay (cAMP Radioimmunoassay kit, Advanced Magnetics,Cambridge, Mass.)

[0092] Tissue Localization Studies. Human tissues (NDRI) werehomogenized and total RNA extracted (Sambrook et al. 1989). cDNA wasprepared from 5 μg of total RNA with random hexanucleotide primers (500pmoles) using Superscript reverse transcriptase (BRL) in PCR reactionbuffer (Cetus Corp.) containing 1 nM dNTPs, at 42° C. for 1 hr. Analiquot of the first strand cDNA was diluted (1:5) in a 50 μl PCRreaction mixture (200 μM dNTPs final concentration) containing 1.25 U ofTaq polymerase and 1 μM of primers from the sense strand(5′TCTATTCTGGAGGCACCAAGGAAC3′) and from the antisense strand(5′TTGTTGATGGGTACGATAAAGATCC3′). The PCR products were run on a 1.5%agarose gel and transferred to charged nylon membrane (ZetaProbe,Bio-Rad). Filters were hybridized and washed under high stringency.

[0093] In Situ Hybridization. In situ hybridization was performed asdescribed previously (McCabe et al., 1989) using male Hartley guineapigs (300-350 g). A fragment of the guinea pig 5-HT_(1F) receptor genewas cloned by homology and sequenced. 45-base oligoprobes synthesized tothe 4,5 loop and 5′ untranslated regions were 3′ end-labeled with355-dATP to a specific activity of 4×10⁹ Ci/mmol. The nucleotidessequences were 5′ GTGATGCGATGATCCACTCATGCTCGCCGTCCCTCGT 3′ and 5′TAGCAGTTCCTCTGAGTCAACTGTTCATAAGAAGAGATTTAGAA 3′. Sense probes, meltingtemperature, and RNase pretreatment were used as controls. Sections wereexposed to Kodak X-OMAT AR film for 1 week or coated with Kodak NTB-2emulsion/2% glycerol(1:1) for 2 weeks. Similar experiments were alsodone on human tissue.

[0094] Drugs: [³H]5-HT (specific activity=28 Ci/mole) was obtained fromNew England Nuclear, Boston, Mass. All other chemicals were obtainedfrom commercial sources and were of the highest grade known purityavailable.

[0095] Results

[0096] Cloning of a Novel Gene Encoding a 5-HT_(1F) Receptor

[0097] Polyadenylated (poly A−) RNA prepared from rat brain was reversetranscribed and the resulting cDNAs were subjected to amplification byPCR with the use of a set of “degenerate” primers. The synthesis ofthese primers were based or sequences corresponding to the third andfifth transmembrane segments of the current set of available serotoninreceptors. The primers were designed to amplify only serotonin specificsequences. This was accomplished, particularly with the transmembranedomain V primer, which was designed to anneal at its end only to hesequence “AFY(F)IP”. We have determined by sequence analysis that thepresence of an alanine (A) rather than a serine (S) in the positionimmediately amino-terminal to the sequence “FY(F)IP” is an amino acidwhich can distinguish the closely related adrenergic and doparinergicreceptor families from the serotonergic receptor family. After 30amplification cycles, agarose (gel electrophoresis revealed a clearpattern of cDNA species of approximately 250 base pairs. IndividualcDNAs were cloned directly into pBluescript and subjected to sequenceanalysis. One clone, designated S51, was observed to encode a novelserotonin receptor. We then screened a human genomic placental librarywith the PCR fragment S51. Isolation of the full-length coding regionwas obtained from a genomic clone designated h116a.

[0098] Nucleotide Sequence and Deduced Amino Acid Sequence of h116a

[0099] DNA sequence information obtained from clone h116a is shown inFIG. 1. An open reading frame extending from an ATG start codon atposition 1 to a stop codon at position 1098 can encode a protein 366amino acids in length, having a relative molecular mass (M_(r)) of41,660. A comparison of this protein sequence with previouslycharacterized neurotransmitter receptors indicates that h116a encodes areceptor which is a new member a family of molecules which span thelipid bilayer seven times and couple to guanine nucleotide regulatoryproteins (the G protein-coupled receptor family). A variety ofstructural features which are invariant in this family were presentincluding the aspartic acid residues of transmembrane regions II andIII, the DRY sequence at the end of transmembrane region III, and theconserved proline residues of transmembrane region IV, V, VI and VII(Hartig et al. and references therein), were present in clone h116a. Acomparison -of the transmembrane homology of h116a to the other clonedserotonin receptors is shown if FIG. 2 exhibits the following order ofidentity: 5-HT_(1Dα) (61%), 5-HT_(1D3) (59%), 5-HT_(1A) (54%), 5HT_(1C)(44%) and 5-HT₂ (44%)

[0100] Receptor Expression in Transfected Mammalian Cell

[0101] Saturation analysis of membranes prepared from stably transfectedLtk⁻ cells demonstrated that the receptor expressed was saturable and ofhigh affinity. Scatchard plot analysis by non-linear regression revealeda Kd of 9.2±0.99 nM (mean±S.E.M., n=4) and a B_(max) 4.4=0.36picomoles/mg of protein (mean±S.E.M., in=4). The percent specificbinding determined at the measured Kd value for [³H]5-HT was greaterthan 85% of total finding. Furthermore, evidence that the receptor iscouple: to a G-protein was demonstrated by the ability of Gpp(NH)p, anon-hydrolyzable analog of GTP, to inhibit the specific binding of[³H]5-HT (IC₅₀=243±115, n_(H)=0.71±0.08, I_(max)=55.6±3.2% ;mean±S.E.M., n=3). Additional data demonstrating that this coupling to aC-protein is functionally relevant :s provided below.

[0102] Pharmacological analysis of the receptor was accomplished bytesting the ability of drugs from different chemical classes to displace[3H]5-HT specific binding (.Table ) of the compounds investigated, 5-HTpossessed the highest affinity which according to the classificationsystem of Peroutka and Snyder (1979) makes this site a member of the5-HT₁ class. Interestingly, 5-CT possessed low affinity and, thus,discriminates this receptor from that of the 5-HT_(1D) receptor as wellas other members of his class. The one exception appears to be therecently cloned 5-HT_(1E) receptor which also has low affinity for 5-CT(U.S. Ser. No. 803,626, filed Dec. 2, 1991, copending). Various ergolinecompounds also bound with high affinity including methylergonovine andmethysergide. Excluding 1-napthylpiperazine (K₁=54), piperazinederivatives had low affinity. Interestingly, the rauwolfia alkaloids,rauwolscine and yohimbine, which are alpha-2 adrenergic antagonists hadfair affinity for this serotonergic receptor. Furthermore, miscellaneousserotonergic agents that possess high affinity for various receptorswithin the serotonin family including ketanserin (5-HT₂), 8-OH-DPAT(5-HT_(1A)), DOI (5-HT_(1C)/5-HT₂), spiperone (5-HT_(1A)/5-HT₂),pindolol (5-HT_(1A)/5-HT_(1B), and zacopride (5-HT₃) had very pooraffinity. Taken together, the pharmacological profile of the 5-HT_(1F)receptor is unique and contrasts to that of other known serotoninreceptors. Interestingly, the agonist rank order of potency (but notantagonist profile) matches one described for large motorneurons in thespinal cord evaluated electrophysiologically (Connel et al., 1989).Accordingly, the probability of developing selective drugs for thisreceptor subtype is increased. The functional 5-HT response (1 μM) wascompletely blocked by the nonselective antagonist methiothepin (10 μM).This antagonism was surmountable (FIG. 3), indicating probablecompetitive antagonism. The dose shift produced by methiothepin yieldedan apparent K_(b) of 438=14 nM, consistent with the K₁ for this compound(Table 1). No direct effect of methiothepin was observed. No othercompound tested in this study was an antagonist. In addition, noevidence for coupling of this receptor to PI turnover was detected at adose of 10 μM 5-HT. TABLE 1 Ki (nM) values of various drugs for theinhibition of [³H]5-HT specific binding to clonal 5-HT_(1F) cellmembranes. Binding assays were performed with 4.5-5.5 nM of [³H]5-HT and10-12 different concentrations of each inhibitory drug. Ki values werecalculated from the IC₅₀ values using the Cheng-Prusoff equation. Eachvalue is the mean ± S.E.M. of 2-4 independent determinations. COMPOUNDKi (nM) 5-HT 10.3 ± 2.0 Sumatriptan  23.0 ± 11.0 Ergonovine 31.0 ± 1.5Methylergonovine  31.0 ± 11.0 Methysergide 34.0 ± 4.9 5-Methoxy-N,N-DMT37.5 ± 1.5 1-Napthylpiperazine 54.0 ± 3.8 Yohimbine  92.0 ± 11.0Ergotamine 171 ± 28 α-Methyl-5-HT 184 ± 35 NAN 190 203 ± 13Dihydroergotamine 276 ± 49 Metergoline 341 ± 71 2-Methyl-5-HT  413 ± 5.6Methiothepin 652 ± 41 5-CT 717 ± 71 TFMPP 1,002 ± 85   5-MT 1,166 ± 197 SCH 23390 1,492 ± 165  5-Benzoxytryptamine 1,495 ± 893  DP-5-CT 1,613 ±817  DOI 1,739 ± 84   8-OH-DPAT 1,772 ± 38   5-Fluorotryptamine 1,805 ±220  mCPP 2,020 ± 36   Tryptamine 2,409 ± 103  Quipazine 4,668 ± 814 Ritanserin 3,521 ± 86   Propanolol 8,706 ± 97   Ketanserin >10,000Spiperone >10,000 Zacopride >10,000 Pindolol >10,000 Mesulergine >10,000Harmaline >10,000 Melatonin >10,000

[0103] cAMP Assay

[0104] Additional supporting evidence that the 5-HT1F receptor isfunctionally coupled to a G-protein was obtained by testing the abilityof 5-HT as well as other representative serotonergic drugs to inhibitforskolin stimulated cAMP production in NIH373 cells transfected withthe 5-HT1F receptor. The endogenous indoleamine, 5-HT, produced aconcentration-related decrease in forskolin-stimulated cAMP productionwith an EC50 of 7.1±1.3 nM (n=4). The maximum inhibition of cAMPproduction by 5-HT was 67±5.4%. Additionally, the serotonergic compounds1-napthylpiperazine and lysergol inhibited forskolin-stimulated cAMPproduction with EC50 values of 4.5±0.2 nM and 8.8±4.3 M (n=2),respectively.

[0105] Receptor Localization Studies

[0106] Expression of the 5-HT_(1F) transcripts was analyzed fromPCR-northern blots and in situ hybridization studies. By PCR, wedetected 5-HT_(1F) receptor mRNA in the human brain, uterus (endometriumand myonetrium) and mesentery (FIG. 4) but not in kidney, liver, spleen,heart, pancreas, or testes. In in situ hybridization experiments, weobserved 5-HT_(1F) transcripts in lamina V of frontal cortex (FIG. 5A)in large pyramidal cells (FIG. 5D). Moderate labeling was also detectedover layer VI non-pyramidal neurons. In both layer V and layer XI, thelabeling was most evident in dorsal sensorimotor neocortex, and incingulate and retrosplenal cortices (FIG. 5C). The pyramidal cells inthe piriform cortex were heavily labeled as were large neurons in theraphe nuclei (FIG. 5E). Hippocampal pyramidal cells in CA1-CA3 weremoderately labeled, as were the granule cells in the dentate gyrus, andsome neurons in the nucleus of the solitary tract. Little labeling wasfound in the thalamus and hypothalamus. Significant labelling was alsofound in the large motoneurons of the ventral horn of the spinal cord.The localization in the human was found to be in good concordance withthat observed in the guinea pig.

[0107] Discussion

[0108] The deduced amino acid sequence of h116a was analyze, to uncoverrelationships between, and the other clones serotonin receptorsequences. Although the homology within the membrane spanning domainswas greatest with the 5-HT_(1Dα) receptor (FIG. 2), the nature of thisnewly cloned receptor could not be clearly predicted. The rational forthis ambiguity is the interpretation of the transmembrane domainhomology (approximately 60%) to he 5-HT_(1Dα) and 5-HT_(1Dβ) receptorsubfamily. Closely related members of a “subfamily” of serotoninreceptors (i.e. “subtypes”) generally share a common transmitter andalso have similar pharmacological profiles and physiological roles (forexample, 5-HT₂ and 5-HT_(1C) or 5-HT_(1Dα) and 5-HT_(1Dβ)). Suchsubtypes” display an amino acid identity of approximately 75-80% intheir transmembrane domains. Serotonin receptors which are no members ofthe same “subfamily”, but are members of the serotonin “family” (inwhich the receptors use the same neurotransmitter; i.e. 5-HT₂ and5-HT_(1Dα)) generally show much lower transmembrane homology(approximately 45%). Such transmembrane amino acid homologous can,therefore, give insight into the relationship between receptors and beused as predictors of receptor pharmacology. According to this type ofanalysis, although the newly cloned receptor appears to be more relatedto the 5-HT_(1D) subfamily, it is likely to be in a subfamily distinctfrom all the other serotonin receptors. Interestingly, the transmembranehomology between the 5-HT_(1E) (Levy et al., 1992; McAllister et al,1992; Zgombick et al., 1992) and 5-HT_(1F) (Amlaiky et al., 1992; Adhanet al., in press) receptors is 72%. It is therefore possible that thesereceptors may be “subtypes”, rather than members of distinct“subfamilies”.

[0109] The present pharmacological evidence substantiates he existenceof a novel serotonin receptor in the human brain and peripheral tissues.Comparison of the binding affinities for various drugs observed innative membranes for other known serotonergic receptors (see Hoyer,1989, to that of the 5-HT_(1F) receptor demonstrates that thepharmacological profile does not fit any known receptor to date. Thecloning of the 5-HT_(1F) site will now allow more extensiveinvestigations into the nature of this unique serotonergic receptor.

[0110] The structure-activity relationships observed in the presentstudy suggest that there are important requirements for high affinitybinding to the 5-HT_(1F) receptor. Substitution or removal of the5-hydroxy group on serotonin significantly decreases the affinity forthe receptor (egs., tryptamine, 5-methoxytryptamine and5-carboxyamidotryptamine). Additionally, α-methylation and 2-methylationof 5-HT lowers its affinity by 20 and 40 fold, respectively, for the5-HT_(1F) site. In contrast to these substitutions, N,N-dimethylation ofthe aliphatic side chain of the indole ring increases the affinityapproximately 20 fold (unpublished observations) Interestingly,5-methoxy-N,N-dimethyltryptamine which possesses both a 5-hydroxysubstitution as well as a N,N-dimethylation has an affinity much higherthan the other 5-substituted tryptamine derivatives. Basic structuralrequirements of the ergoline derivatives demonstrate that N-methylationof the indole ring does not decrease affinity as does bulkssubstitutions. Furthermore, piperazine derivatives are not bound at highaffinity.

[0111] Notably, the application of the human 5-HT_(1F) receptor clone topharmaceutical research can lead to new drug design and development. Inthis regard, it is important to point out that the affinities ofsumatriptan, methylergonovine and methysergide for this receptor suggestthat this site may be involved in the control of migraine headaches.Certainly, these compounds have had success in the clinic for thetreatment of this debilitating disorder (Sleight et al., 1990). Notably,however, it has been thought that the action of these compounds ismediated at 5-HT1D receptors for sumatriptan and 5-HT2 receptors formethysergide. Interestingly, methylergonovine may be an activemetabolite of methysergide which can be responsible for some of thetherapeutic antimigraine effects of methysergide. This novel site withaffinity for these agents Would now suggest that there is oneserotonergic receptor which may be responsible for both the pathogenesisand, accordingly the pharmacological treatment. Importantly, the agentsdescribed for migraine are not selective for any one particularserotonin receptor and, thus, the physiological significance of drugsacting at one specific site remains controversial (Humphrey P. P. A. etal., 1990). The notion that the 5-HT_(1F) receptor is involved inmigraine may be supported by evidence demonstrating that metergolinewhich has high affinity for the 5-HT_(1F) receptor does not block theeffects of sumatriptan in the dog saphenous vein (Sumner and Humphrey,1990) inferring that this vascular model mad contain the novel 5-HT_(1F)site. Furthermore, this data can support the idea that sumatriptan actsat 5-HT_(1F) receptors as an anti-migraine drug. Localization oftranscripts for the 5-HT_(1F) receptor in the spinal trigeminal nucleusby in situ hybridization strongly supports this contention (Buzzi etal.,1990, 1991; Moskowitz et al., 1992). The potential of the 5-HT_(1F)receptor as a novel target for migraine where selective drugs may bedeveloped is an exciting possibility which needs to be explored.

[0112] Further insight into potential therapeutic significance of the5-HT1F receptor has been obtained through localization studies using PCRand in situ hybridization. Localization of transcripts for this receptorindicates a relatively selective tissue distribution. Of tissuesreported here, the 5-HT_(1F) receptor was only detected in a fewincluding the brain, uterus, and mesentery. The possible role of thisreceptor in uterine or vascular function is intriguing. Future studiesdefining the specific cell type(s) in these tissues which express thereceptor may provide insight into its function in the periphery.Possibilities for therapeutic benefit include dysmenorrhea and laborinduction uterus) and hypertension (vascular components of mesentery)and obesity (adipose components). In the brain, the expression of the5-HT1F receptor had a limited distribution compared to that of otherserotonin receptors. In the neocortex, labelling of layer V pyramidalneurons may indicate a functional role for the 5-HT_(1F) receptorprotein in the integration of sensorimotor (somatodendritic; frontalcortex) or afferent information associated with limbic functions(somatodendritic; cingulate/retrosplenial cortex), or in spinal cordprocesses (axonal). Intense labeling was detected in the largemotoneurons of the ventral horn of the spinal cord. Strong labeling wasalso detected in hippocampal pyramidal cells, in several thalamicnuclei, and in the dorsal raphe. The detection of transcripts for thisgene in the dorsal raphe nucleus indicates a possible role as anautoreceptor. Autoreceptor function opens the possibility that the5-HT_(1F) receptor could be involved in any or all of the known actionsof serotonin including therapeutic potential in anxiety, depression,sleep disorders including )et lag, appetite control, sexual dysfunction,gastrointestinal motility including irritable bowel disease, andcardiovascular regulation. In addition, localization to the largemotoneurons indicates a possible role in spasticity and other disordersof movement.

[0113] Another consideration for therapeutic application of this sitemay be related to the treatment of feeding disorders such as obesity,bulimea nervosa and/or anorexia nervosa. The involvement of serotoninand feeding behavior has received much attention during the last decade.It is now known that many of the identified and well-characterizedserotonin receptors are capable of modulating feeding (Blundell andLawton, 1990). Notably, serotonin uptake blockers which have seen usedto treat feeding disorders act nonselectively and as such haveside-effect potential (Jimerson et al., 1990). The fact that the5-HT_(1F) receptor has been cloned from both peripheral and centralsites, and has been localized by both PCR and by in situ hybridization,suggests from an anatomical standpoint that it can be found in strategiclocations where feeding may be altered. Although many differentserotonergic receptors are involved in feeding, the search for the onesite that can be exploited for selective drug development has yet to befound. There is no doubt that interest exists in finding drugs thatinteract with the serotonin system for the treatment of feedingdisorders (Cooper, 1989;

[0114] Overall, the 5-HT_(1F) receptor can be an important sitestimulated by nonselectively blocking serotonin uptake as isaccomplished with certain antidepressants. In regard to this, serotoninuptake blockers are effective in treating neuropsychiatric disorderssuch as depression and obsessive-compulsive illness (Asberg et al.,1986; Sleight et al., 1990: Insel et al., 1985). However, these agentshave side effects and, in fact, the mechanism of action for thesecompounds are not linked to any particular serotonergic receptor. Thepossibility that agents selective for the 5-HT_(1F) receptor may haveclinical utility as antidepressants, for example, without the sideeffects attributed to current treatment modalities can have significantimplications for drug therapy. The localization of the 5-HT1F receptorin the raphe nuclei, and therefore its potential role as anautoreceptor, further supports the role for this receptor subtype indepression.

[0115] In summary, the pharmacological profile of the clones human5-HT_(1F) receptor s unique and contrasts to other known serotonergicreceptors. The utility of this site expressed in a cellular system and,thus, isolated for study will create excellent opportunities in drugdevelopment directed towards a novel serotonergic receptor that may havewide-range implications for drug therapy. Ultimately, indepthinvestigations onto the localization of this receptor in brain andperipheral tissue will target new sites that may lead to functionalroles of the serotonergic receptor. Indeed, the potential therapeuticapplications may extend to neuropsychiatric disorders includingdepression, anxiety, schizophrenia, dementia and obsessive-compulsiveillness as well as obesity and migraine.

[0116] Additionally, the localization of the 5-HT_(1F) receptor in thespinal cord suggests possible roles for this subtype in analgesia aswell as spasticity. The clear evidence of involvement of this receptorin the ventral horn further supports the possible role in motor control.Interestingly, the agonist profile of the 5-HT_(1F) receptor matchesthat reported for large motoneurons of the spinal cord measuredelectrophysiologically (Connel et al., 1989). In addition, the presenceof the 5-HT_(1F) receptor in the mesentery, at major resistance bed ofthe vascular tree, indicated a role in the control of blood pressure. Adetailed accounting of the localization and therapeutic potential ispresented in Table II. TABLE II Summary of the localization of mRNA forthe 5-HT_(1F) receptor in the guinea pig and human CNS. Experiments wereperformed as described (methods). Each experiment was replicated 2-3times. Potential therapeutic roles anticipated base on these data areindicated. LOCALIZATION OF HUMAN 5HT_(1F) mRNA* THERAPEUTIC AREASPROJECTIONS RELEVANCE FRONTAL CORTEX Main projections to Potentialapplication striatum, dorsal for the development thalamus, and oftreatments for superior colliculus. schizophrenia and mood disorders.CAUDATE Primary projections Potential treatment NUCLEUS to globuspallidus, of any basal ganglia substantia nigra. disorder, includingParkinson's disease, Huntington's chorea, or tardive dyskinesia.HIPPOCAMPAL Pyramidal neurons Primary locus for FORMATION project mainlytreatment of memory within the disorders, e.g. hippocampus, andAlzheimer's disease also to the septum. or for cognitive enhancement inpeople with learning disabilities. Also possible treatment for temporallobe epilepsy. AMYGDALA Cells in amygdala Wide range of have widespreadpotential projections to applications. These cortex, hippocampus,include treatment of basal ganglia, autonomic hypothalamus, anddysfunctions such as brainstem autonomic cardiac arrhythmias centers.and non-adaptive response to environmental stressors. Also potentialtreatment of mood disorders, such as bipolar syndrome. HYPOGLOSSAL Mainprojections to Treatment of verbal NUCLEUS somatic skeletal apraxia.musculature of the tongue. DORSAL Principal May have some EFFERENTprojections are to application to the NUCLEUS OF THE the parasympathetictreatment of stress- VAGUS ganglia and related ulcers and abdominalviscera. iritable bowel disease. NUCLEUS OF THE Main projections areVaried potential SOLITARY TRACT to thalamus, applications, withamygdala, regulation of cardio- rostroventral vascular function themedulla, and the Al most prominent, e.g. noradrenergic cell ananti-hypertensive. group of the dorsal medulla. GRACILE Providesinnervation Potential NUCLEUS of lumbosacral applications for the spinalcord. treatment of dermatitis, or pain associated with itching. CUNEATEProvides innervation Potential NUCLEUS of cervical spinal applicationsfor the cord. treatment of dermatitis, or pain associated with itching.SPINAL Main projections are Potential treatment TRIGEMINAL to thecontralateral of migraine NUCLEUS ventrobasal headaches, and otherthalamus, the pain syndromes such posterior thalamic as trigeminal n.,the zona neuralgia. incerta, the superior colliculus, and the motornuclei of trigeminal. OLIVARY Primary projections Treatment of ataxiaCOMPLEX are to the associated with cerebellum. olivopontocerebellaratrophy, or tremors accompanying some neurodegenerative diseasesRETICULAR Projections to the Involvement in FORMATION intra-laminar andcardiac pressor and dorsomedial n. of depressor responses thalamus, thesuggests a role in hypothalamus, blood pressure supramammillary andregulation and lateral mammillary possibly a treatment nuclei,theseptum, for hypertension. the diagonal band, Also possible spinalcord, application for the cerebellum, treatment of urinary brainstemautonomic retention disorders, nuclei. and in the management of pain.MEDIAL Projections to Treatment of motion VESTIBULAR oculomotor complexsickness. NUCLEUS and cervical spinal cord rotor neurons. CEREBELLARProjections only to Potential treatment PURKINJE CELLS deep cerebellarof movement nuclei. disorders, particularly those involving plannedmovements, or those invloving abnormalities of gait or stance. SPINALCORD Ascending dorsal Primary site for VENTRAL HORN horn projections totreatment of pain, thalamus, brainstem and for possible reticularformation anesthetic and central gray. applications. Also Ventral hornpossible therapies projections to for spasticity and skeletal and/ormovement disorders. smooth muscle. ANTERIOR Widespread Treatment ofOLFACTORY projections to olfactory disorders NUCLEI and brain olfactory(dysosmias) PIRIFORM centers, to limbic associated with many CORTEXsystem, neurological hypothalamus, syndromes. thalamus, and striatum.LAYER V of Cells of layer V Enhancement of memory NEOCORTEX projectprimarily for motor tasks, to other cortical particularly in areas, andto certain amnestic basal ganglia. syndromes, e.g. Alzheimer's disease.CAUDATE- Medium spiny Potential treatment PUTAMEN and neurons project toof any basal ganglia NUCLEUS globus pallidus, disorder, includingACCUMBENS entopeduncular nl, Parkinson's disease, and substantiaHuntington's chorea, nigra. or tardive dyskinesia. AMYGDALA Cells inamygdala Wide range of have widespread potential projections toapplications. These cortex, include treatment of hippocampus, basalautonomic ganglia, dysfunctions such as hypothalamus, and cardiacarrhythmias brainstem and non-adaptive autonomic centers. response toenvironmental stressors. Also potential treatment of mood disorders,such as bipolar syndrome. HIPPOCAMPUS Pyramidal neurons Primary locusfor project mainly treatment of memory within the disorders, e.g.hippocampus, and Alzheimer's disease also to the or for cognitiveseptum. enhancement in people with learning disabilities. Also possibletreatment for temporal lobe epilepsy. DORSAL RAPHE Extensive Treatmentof pain projections to syndromes, including cerebral cortex, migraineheadache. frontal striatum, Involvement of raphe limbic structures, ingeneral olfactory arousal/attentional tubercle, central processes makesthis gray, hippocampus, a possible target for and spinal cord. treatmentof attentional dysfunctions, such as those observed in Alzheimer'sdisease, or in developmental disabilities. Potential application in thetreatment of depression. PONTINE Major projection Potential treatmentNUCLEI is to the of movement cerebellar cortex. disorders, particularlyplanned movement, and gait disorders such as Friedrich's ataxia.INFERIOR Major obligatory COLLICULUS synaptic station in ascendingauditory pathway. TRIGEMINAL Main projections Potential treatmentNUCLEAR are to the of migraine COMPLEX contralateral headaches, andother ventrobasal pain syndromes such thalamus, the as trigeminalposterior thalamic neuralgia. n., the zona incerta, the superiorcolliculus, and the motor nuclei of trigeminal. PONTINE Projections tothe Involvement in RETICULAR intra-laminar and cardiac pressor andFORMATION dorsomedial n. of depressor responses A. GIGANTOCEL- thalamus,the suggests a role in LULAR hypothalamus, blood pressure RETICULARsupramammillary regulation and NUCLEUS and lateral possibly a treatmentB. PARAGIGANT mammillary nuclei, for hypertension. OCELLULAR the Alsopossible RETICULAR septum, the application for the NUCLEUS diagonalband, treatment of urinary C. RAPHE spinal cord, retention disorders,MAGNUS cerebellum, and in the management brainstem of pain. autonomicnuclei MEDIAL Projections to Treatment of motion VESTIBULAR oculomotorcomplex sickness. NUCLEUS and cervical spinal cord motor neurons.CEREBELLAR Projections only Potential treatment PURKINJE to deepcerebellar of movement CELLS nuclei. disorders, particularly thoseinvolving planned movements, or those invloving abnormalities of gait orstance. SPINAL CORD Ascending dorsal Primary site for horn projectionstreatment of pain, to thalamus, and for possible brainstem anestheticreticular applications. Also formation and possible therapies centralgray. for spasticity and Ventral horn movement disorders. projections toskeletal and/or smooth muscle.

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1 13 1 1730 DNA Homo sapiens 1 ttgcatgcct gcaggtcgac tctagaggatccccgggtac cgagctcgaa ttcctttgtt 60 attttgtcat gcttcaagcc taggaaaagcctaagcaaaa ctcttggtgg gctctttgtt 120 acattccagc ctttgaataa gggcactggctctatcagct ttgaatatat aactcaacta 180 gtcagtcagt agtactgaaa cagttgttacggaggcctgc gttattgaga tcgggcctgc 240 cacactttta aactttttct gacatggacaaagagaaaaa ccaattctat aatggcagag 300 atttcactga gtaacaagct agagtatcattaaaaattgt tgtatttaac ctatatttta 360 agaaatgttt tggaagttac tggctttttttactgttctc attaaatttc ttaaataaaa 420 aggaaaacta aaaccttcaa tctgaacctcatttttttaa tctatagaat attctgggta 480 aacataacat acacttttta aaaattattctgaaaggaag agaaaagttc ttgaagcctt 540 ctctgaactg ttttttctct tcccttgttacaggtatcca tttttcagct atattaatct 600 tttaaaacaa agaaaatgga tttcttaaattcatctgatc aaaacttgac ctcagaggaa 660 ctgttaaaca gaatgccatc caaaattctggtgtccctca ctctgtctgg gctggcactg 720 atgacaacaa ctatcaactc ccttgtgatcgctgcaatta ttgtgacccg gaagctgcac 780 catccagcca attatttaat ttgttcccttgcagtcacag attttcttgt ggctgtcctg 840 gtgatgccct tcagcattgt gtatattgtgagagagagct ggattatggg gcaagtggtc 900 tgtgacattt ggctgagtgt tgacattacctgctgcacgt gctccatctt gcatctctca 960 gctatagctt tggatcggta tcgagcaatcacagatgctg ttgagtatgc caggaaaagg 1020 actccaaagc atgctggcat tatgattacaatagtttgga ttatatctgt ttttatctct 1080 atgcctcctc tattctggag gcaccaaggaactagcagag atgatgaatg catcatcaag 1140 cacgaccaca ttgtttccac catttactcaacatttggag ctttctacat cccactggca 1200 ttgattttga tcctttacta caaaatatatagagcagcaa agacattata ccacaagaga 1260 caagcaagta ggattgcaaa ggaggaggtgaatggccaag tccttttgga gagtggtgag 1320 aaaagcacta aatcagtttc cacatcctatgtactagaaa agtctttatc tgacccatca 1380 acagactttg ataaaattca tagcacagtgagaagtctca ggtctgaatt caagcatgag 1440 aaatcttgga gaaggcaaaa gatctcaggtacaagagaac ggaaagcagc cactaccctg 1500 ggattaatct tgggtgcatt tgtaatatgttggcttcctt tttttgtaaa agaattagtt 1560 gttaatgtct gtgacaaatg taaaatttctgaagaaatgt ccaatttttt ggcatggctt 1620 gggtatctca attcccttat aaatccactgatttacacaa tctttaatga agacttcaag 1680 aaagcattcc aaaagcttgt gcgatgtcgatgttagtttt aaaaatgttt 1730 2 366 PRT Homo sapiens 2 Met Asp Phe Leu AsnSer Ser Asp Gln Asn Leu Thr Ser Glu Glu Leu 1 5 10 15 Leu Asn Arg MetPro Ser Lys Ile Leu Val Ser Leu Thr Leu Ser Gly 20 25 30 Leu Ala Leu MetThr Thr Thr Ile Asn Ser Leu Val Ile Ala Ala Ile 35 40 45 Ile Val Thr ArgLys Leu His His Pro Ala Asn Tyr Leu Ile Cys Ser 50 55 60 Leu Ala Val ThrAsp Phe Leu Val Ala Val Leu Val Met Pro Phe Ser 65 70 75 80 Ile Val TyrIle Val Arg Glu Ser Trp Ile Met Gly Gln Val Val Cys 85 90 95 Asp Ile TrpLeu Ser Val Asp Ile Thr Cys Cys Thr Cys Ser Ile Leu 100 105 110 His LeuSer Ala Ile Ala Leu Asp Arg Tyr Arg Ala Ile Thr Asp Ala 115 120 125 ValGlu Tyr Ala Arg Lys Arg Thr Pro Lys His Ala Gly Ile Met Ile 130 135 140Thr Ile Val Trp Ile Ile Ser Val Phe Ile Ser Met Pro Pro Leu Phe 145 150155 160 Trp Arg His Gln Gly Thr Ser Arg Asp Asp Glu Cys Ile Ile Lys His165 170 175 Asp His Ile Val Ser Thr Ile Tyr Ser Thr Phe Gly Ala Phe TyrIle 180 185 190 Pro Leu Ala Leu Ile Leu Ile Leu Tyr Tyr Lys Ile Tyr ArgAla Ala 195 200 205 Lys Thr Leu Tyr His Lys Arg Gln Ala Ser Arg Ile AlaLys Glu Glu 210 215 220 Val Asn Gly Gln Val Leu Leu Glu Ser Gly Glu LysSer Thr Lys Ser 225 230 235 240 Val Ser Thr Ser Tyr Val Leu Glu Lys SerLeu Ser Asp Pro Ser Thr 245 250 255 Asp Phe Asp Lys Ile His Ser Thr ValArg Ser Leu Arg Ser Glu Phe 260 265 270 Lys His Glu Lys Ser Trp Arg ArgGln Lys Ile Ser Gly Thr Arg Glu 275 280 285 Arg Lys Ala Ala Thr Thr LeuGly Leu Ile Leu Gly Ala Phe Val Ile 290 295 300 Cys Trp Leu Pro Phe PheVal Lys Glu Leu Val Val Asn Val Cys Asp 305 310 315 320 Lys Cys Lys IleSer Glu Glu Met Ser Asn Phe Leu Ala Trp Leu Gly 325 330 335 Tyr Leu AsnSer Leu Ile Asn Pro Leu Ile Tyr Thr Ile Phe Asn Glu 340 345 350 Asp PheLys Lys Ala Phe Gln Lys Leu Val Arg Cys Arg Cys 355 360 365 3 422 PRTHomo sapiens 3 Met Asp Val Leu Ser Pro Gly Gln Gly Asn Asn Thr Thr SerPro Pro 1 5 10 15 Ala Pro Phe Glu Thr Gly Gly Asn Thr Thr Gly Ile SerAsp Val Thr 20 25 30 Val Ser Tyr Gln Val Ile Thr Ser Leu Leu Leu Gly ThrLeu Ile Phe 35 40 45 Cys Ala Val Leu Gly Asn Ala Cys Val Val Ala Ala IleAla Leu Glu 50 55 60 Arg Ser Leu Gln Asn Val Ala Asn Tyr Leu Ile Gly SerLeu Ala Val 65 70 75 80 Thr Asp Leu Met Val Ser Val Leu Val Leu Pro MetAla Ala Leu Tyr 85 90 95 Gln Val Leu Asn Lys Trp Thr Leu Gly Gln Val ThrCys Asp Leu Phe 100 105 110 Ile Ala Leu Asp Val Leu Cys Cys Thr Ser SerIle Leu His Leu Cys 115 120 125 Ala Ile Ala Leu Asp Arg Tyr Trp Ala IleThr Asp Pro Ile Asp Tyr 130 135 140 Val Asn Lys Arg Thr Pro Arg Arg AlaAla Ala Leu Ile Ser Leu Thr 145 150 155 160 Trp Leu Ile Gly Phe Leu IleSer Ile Pro Pro Met Leu Gly Trp Arg 165 170 175 Thr Pro Glu Asp Arg SerAsp Pro Asp Ala Cys Thr Ile Ser Lys Asp 180 185 190 His Gly Tyr Thr IleTyr Ser Thr Phe Gly Ala Phe Tyr Ile Pro Leu 195 200 205 Leu Leu Met LeuVal Leu Tyr Gly Arg Ile Phe Arg Ala Ala Arg Phe 210 215 220 Arg Ile ArgLys Thr Val Lys Lys Val Glu Lys Thr Gly Ala Asp Thr 225 230 235 240 ArgHis Gly Ala Ser Pro Ala Pro Gln Pro Lys Lys Ser Val Asn Gly 245 250 255Glu Ser Gly Ser Arg Asn Trp Arg Leu Gly Val Glu Ser Lys Ala Gly 260 265270 Gly Ala Leu Cys Ala Asn Gly Ala Val Arg Gln Gly Asp Asp Gly Ala 275280 285 Ala Leu Glu Val Ile Glu Val His Arg Val Gly Asn Ser Lys Glu His290 295 300 Leu Pro Leu Pro Ser Glu Ala Gly Pro Thr Pro Cys Ala Pro AlaSer 305 310 315 320 Phe Glu Arg Lys Asn Glu Arg Asn Ala Glu Ala Lys ArgLys Met Ala 325 330 335 Leu Ala Arg Glu Arg Lys Thr Val Lys Thr Leu GlyIle Ile Met Gly 340 345 350 Thr Phe Ile Leu Cys Trp Leu Pro Phe Phe IleVal Ala Leu Val Leu 355 360 365 Pro Phe Cys Glu Ser Ser Cys His Met ProThr Leu Leu Gly Ala Ile 370 375 380 Ile Asn Trp Leu Gly Tyr Ser Asn SerLeu Leu Asn Pro Val Ile Tyr 385 390 395 400 Ala Tyr Phe Asn Lys Asp PheGln Asn Ala Phe Lys Lys Ile Ile Lys 405 410 415 Cys Leu Phe Cys Arg Gln420 4 460 PRT Homo sapiens 4 Met Val Asn Leu Gly Asn Ala Val Arg Ser LeuLeu Met His Leu Ile 1 5 10 15 Gly Leu Leu Val Trp Gln Phe Asp Ile SerIle Ser Pro Val Ala Ala 20 25 30 Ile Val Thr Asp Thr Phe Asn Ser Ser AspGly Gly Arg Leu Phe Gln 35 40 45 Phe Pro Asp Gly Val Gln Asn Trp Pro AlaLeu Ser Ile Val Val Ile 50 55 60 Ile Ile Met Thr Ile Gly Gly Asn Ile LeuVal Ile Met Ala Val Ser 65 70 75 80 Met Glu Lys Lys Leu His Asn Ala ThrAsn Tyr Phe Leu Met Ser Leu 85 90 95 Ala Ile Ala Asp Met Leu Val Gly LeuLeu Val Met Pro Leu Ser Leu 100 105 110 Leu Ala Ile Leu Tyr Asp Tyr ValTrp Pro Leu Pro Arg Tyr Leu Cys 115 120 125 Pro Val Trp Ile Ser Leu AspVal Leu Phe Ser Thr Ala Ser Ile Met 130 135 140 His Leu Cys Ala Ile SerLeu Asp Arg Tyr Val Ala Ile Arg Asn Pro 145 150 155 160 Ile Glu His SerArg Phe Asn Ser Arg Thr Lys Ala Ile Met Lys Ile 165 170 175 Ala Ile ValTrp Ala Ile Ser Ile Gly Val Ser Val Pro Ile Pro Val 180 185 190 Ile GlyLeu Arg Asp Glu Ser Lys Val Phe Val Asn Asn Thr Thr Cys 195 200 205 ValLeu Asn Asp Pro Asn Phe Val Leu Ile Gly Ser Phe Val Ala Phe 210 215 220Phe Ile Pro Leu Thr Ile Met Val Ile Thr Tyr Phe Leu Thr Ile Tyr 225 230235 240 Val Leu Arg Arg Gln Thr Leu Met Leu Leu Arg Gly His Thr Glu Glu245 250 255 Glu Leu Ala Asn Met Ser Leu Asn Phe Leu Asn Cys Cys Cys LysLys 260 265 270 Asn Gly Gly Glu Glu Glu Asn Ala Pro Asn Pro Asn Pro AspGln Lys 275 280 285 Pro Arg Arg Lys Lys Lys Glu Lys Arg Pro Arg Gly ThrMet Gln Ala 290 295 300 Ile Asn Asn Glu Lys Lys Ala Ser Lys Val Leu GlyIle Val Phe Phe 305 310 315 320 Val Phe Leu Ile Met Trp Cys Pro Phe PheIle Thr Asn Ile Leu Ser 325 330 335 Val Leu Cys Gly Lys Ala Cys Asn GlnLys Leu Met Glu Lys Leu Leu 340 345 350 Asn Val Phe Val Trp Ile Gly TyrVal Cys Ser Gly Ile Asn Pro Leu 355 360 365 Val Tyr Thr Leu Phe Asn LysIle Tyr Arg Arg Ala Phe Ser Lys Tyr 370 375 380 Leu Arg Cys Asp Tyr LysPro Asp Lys Lys Pro Pro Val Arg Gln Ile 385 390 395 400 Pro Arg Val AlaAla Thr Ala Leu Ser Gly Arg Glu Leu Asn Val Asn 405 410 415 Ile Tyr ArgHis Thr Asn Glu Arg Val Ala Arg Lys Ala Asn Asp Pro 420 425 430 Glu ProGly Ile Glu Met Gln Val Glu Asn Leu Glu Leu Pro Val Asn 435 440 445 ProSer Asn Val Val Ser Glu Arg Ile Ser Ser Val 450 455 460 5 376 PRT Homosapiens 5 Met Ser Pro Leu Asn Gln Ser Ala Glu Gly Leu Pro Gln Glu AlaSer 1 5 10 15 Asn Arg Ser Leu Asn Ala Thr Glu Thr Ser Glu Ala Trp AspPro Arg 20 25 30 Thr Leu Gln Ala Leu Lys Ile Ser Leu Ala Val Val Leu SerVal Ile 35 40 45 Thr Leu Ala Thr Val Leu Ser Asn Ala Phe Val Leu Thr ThrIle Leu 50 55 60 Leu Thr Arg Lys Leu His Thr Pro Ala Asn Tyr Leu Ile GlySer Leu 65 70 75 80 Ala Thr Thr Asp Leu Leu Val Ser Ile Leu Val Met ProIle Ser Met 85 90 95 Ala Tyr Thr Ile Thr His Thr Trp Asn Phe Gly Gln IleLeu Cys Asp 100 105 110 Ile Trp Leu Ser Ser Asp Ile Thr Cys Cys Thr AlaSer Ile Leu His 115 120 125 Leu Cys Val Ile Ala Leu Asp Arg Tyr Trp AlaIle Thr Asp Ala Leu 130 135 140 Glu Tyr Ser Lys Arg Arg Thr Ala Gly HisAla Ala Thr Met Ile Ala 145 150 155 160 Ile Val Trp Ala Ile Ser Ile CysIle Ser Ile Pro Pro Leu Phe Trp 165 170 175 Arg Gln Ala Lys Ala Gln GluGlu Met Ser Asp Cys Leu Val Asn Thr 180 185 190 Ser Gln Ile Ser Tyr ThrIle Tyr Ser Thr Cys Gly Ala Phe Tyr Ile 195 200 205 Pro Ser Val Leu LeuIle Ile Leu Tyr Gly Arg Ile Tyr Arg Ala Ala 210 215 220 Arg Asn Arg IleLeu Asn Pro Pro Ser Leu Tyr Gly Lys Arg Phe Thr 225 230 235 240 Thr AlaHis Leu Ile Thr Gly Ser Gly Ser Ser Leu Cys Ser Leu Asn 245 250 255 SerSer Leu His Glu Gly His Ser His Ser Ala Gly Ser Pro Leu Phe 260 265 270Phe Asn His Val Lys Ile Lys Leu Ala Asp Ser Ala Leu Glu Arg Lys 275 280285 Arg Ile Ser Ala Ala Arg Glu Arg Lys Ala Thr Lys Ile Leu Gly Ile 290295 300 Ile Leu Gly Ala Phe Ile Ile Cys Trp Leu Pro Phe Phe Val Val Ser305 310 315 320 Leu Val Leu Pro Ile Cys Arg Asp Ser Cys Trp Ile His ProGly Leu 325 330 335 Phe Asp Phe Phe Thr Trp Leu Gly Tyr Leu Asn Ser LeuIle Asn Pro 340 345 350 Ile Ile Tyr Thr Val Phe Asn Glu Glu Phe Arg GlnAla Phe Gln Lys 355 360 365 Ile Val Pro Phe Arg Lys Ala Ser 370 375 6390 PRT Homo sapiens 6 Met Glu Glu Pro Gly Ala Gln Cys Ala Pro Pro ProPro Ala Gly Ser 1 5 10 15 Glu Thr Trp Val Pro Gln Ala Asn Leu Ser SerAla Pro Ser Gln Asn 20 25 30 Cys Ser Ala Lys Asp Tyr Ile Tyr Gln Asp SerIle Ser Leu Pro Trp 35 40 45 Lys Val Leu Leu Val Met Leu Leu Ala Leu IleThr Leu Ala Thr Thr 50 55 60 Leu Ser Asn Ala Phe Val Ile Ala Thr Val TyrArg Thr Arg Lys Leu 65 70 75 80 His Thr Pro Ala Asn Tyr Leu Ile Ala SerLeu Ala Val Thr Asp Leu 85 90 95 Leu Val Ser Ile Leu Val Met Pro Ile SerThr Met Tyr Thr Val Thr 100 105 110 Gly Arg Trp Thr Leu Gly Gln Val ValCys Asp Phe Trp Leu Ser Ser 115 120 125 Asp Ile Thr Cys Cys Thr Ala SerIle Leu His Leu Cys Val Ile Ala 130 135 140 Leu Asp Arg Tyr Trp Ala IleThr Asp Val Ala Glu Tyr Ser Ala Lys 145 150 155 160 Arg Thr Pro Lys ArgAla Ala Val Met Ile Ala Leu Val Trp Val Phe 165 170 175 Ser Ile Ser IleSer Leu Pro Pro Phe Phe Trp Arg Gln Ala Lys Ala 180 185 190 Glu Glu GluVal Ser Glu Cys Val Val Asn Thr Asp His Ile Leu Tyr 195 200 205 Thr ValTyr Ser Thr Val Gly Ala Phe Tyr Phe Pro Thr Leu Leu Leu 210 215 220 IleAla Leu Tyr Gly Arg Ile Tyr Val Glu Ala Arg Ser Arg Ile Leu 225 230 235240 Lys Gln Thr Pro Asn Arg Thr Gly Lys Arg Leu Thr Arg Ala Gln Leu 245250 255 Ile Thr Asp Ser Pro Gly Ser Thr Ser Ser Val Thr Ser Ile Asn Ser260 265 270 Arg Val Pro Asp Val Pro Ser Glu Ser Gly Ser Pro Val Tyr ValAsn 275 280 285 Gln Val Lys Val Arg Val Ser Asp Ala Leu Leu Glu Lys LysLys Leu 290 295 300 Met Ala Ala Arg Glu Arg Lys Ala Thr Lys Thr Leu GlyIle Ile Leu 305 310 315 320 Gly Ala Phe Ile Val Cys Trp Leu Pro Phe PheIle Ile Ser Leu Val 325 330 335 Met Pro Ile Cys Lys Asp Ala Cys Trp PheHis Leu Ala Ile Phe Asp 340 345 350 Phe Phe Thr Trp Leu Gly Tyr Leu AsnLeu Ile Asn Pro Ile Ile Tyr 355 360 365 Thr Met Ser Asn Glu Asp Phe LysGln Ala Phe His Lys Leu Ile Arg 370 375 380 Phe Ile Cys Cys Thr Ser 385390 7 366 PRT Homo sapiens 7 Met Asp Phe Leu Asn Ser Ser Asp Gln Asn LeuThr Ser Glu Glu Leu 1 5 10 15 Leu Asn Arg Met Pro Ser Lys Ile Leu ValSer Leu Thr Leu Ser Gly 20 25 30 Leu Ala Leu Met Thr Thr Thr Ile Asn SerLeu Val Ile Ala Ala Ile 35 40 45 Ile Val Thr Arg Lys Leu His His Pro AlaAsn Tyr Leu Ile Cys Ser 50 55 60 Leu Ala Val Thr Asp Phe Leu Val Ala ValLeu Val Met Pro Phe Ser 65 70 75 80 Ile Val Tyr Ile Val Arg Glu Ser TrpIle Met Gly Gln Val Val Cys 85 90 95 Asp Ile Trp Leu Ser Val Asp Ile ThrCys Cys Thr Cys Ser Ile Leu 100 105 110 His Leu Ser Ala Ile Ala Leu AspArg Tyr Arg Ala Ile Thr Asp Ala 115 120 125 Val Glu Tyr Ala Arg Lys ArgThr Pro Lys His Ala Gly Ile Met Ile 130 135 140 Thr Ile Val Trp Ile IleSer Val Phe Ile Ser Met Pro Pro Leu Phe 145 150 155 160 Trp Arg His GlnGly Thr Ser Arg Asp Asp Glu Cys Ile Ile Lys His 165 170 175 Asp His IleVal Ser Thr Ile Tyr Ser Thr Phe Gly Ala Phe Tyr Ile 180 185 190 Pro LeuAla Leu Ile Leu Ile Leu Tyr Tyr Lys Ile Tyr Arg Ala Ala 195 200 205 LysThr Leu Tyr His Lys Arg Gln Ala Ser Arg Ile Ala Lys Glu Glu 210 215 220Val Asn Gly Gln Val Leu Leu Glu Ser Gly Glu Lys Ser Thr Lys Ser 225 230235 240 Val Ser Thr Ser Tyr Val Leu Glu Lys Ser Leu Ser Asp Pro Ser Thr245 250 255 Asp Phe Asp Lys Ile His Ser Thr Val Arg Ser Leu Arg Ser GluPhe 260 265 270 Lys His Glu Lys Ser Trp Arg Arg Gln Lys Ile Ser Gly ThrArg Glu 275 280 285 Arg Lys Ala Ala Thr Thr Leu Gly Leu Ile Leu Gly AlaPhe Val Ile 290 295 300 Cys Trp Leu Pro Phe Phe Val Lys Glu Leu Val ValAsn Val Cys Asp 305 310 315 320 Lys Cys Lys Ile Ser Glu Glu Met Ser AsnPhe Leu Ala Trp Leu Gly 325 330 335 Tyr Leu Asn Ser Leu Ile Asn Pro LeuIle Tyr Thr Ile Phe Asn Glu 340 345 350 Asp Phe Lys Lys Ala Phe Gln LysLeu Val Arg Cys Arg Cys 355 360 365 8 470 PRT Homo sapiens 8 Met Asp IleLeu Cys Glu Glu Asn Thr Ser Leu Ser Ser Thr Thr Asn 1 5 10 15 Ser LeuMet Gln Leu Asn Asp Asp Thr Arg Leu Tyr Ser Asn Asp Phe 20 25 30 Asn SerGly Glu Ala Asn Thr Ser Asp Ala Phe Asn Trp Thr Val Asp 35 40 45 Ser GluAsn Arg Thr Asn Leu Ser Cys Glu Gly Cys Leu Ser Pro Ser 50 55 60 Cys LeuSer Leu Leu His Leu Gln Glu Lys Asn Trp Ser Ala Leu Leu 65 70 75 80 ThrAla Val Val Ile Ile Leu Thr Ile Ala Gly Asn Ile Leu Val Ile 85 90 95 MetAla Val Ser Leu Glu Lys Lys Leu Gln Asn Ala Thr Asn Tyr Phe 100 105 110Leu Met Ser Leu Ala Ile Ala Asp Met Leu Leu Gly Phe Leu Val Met 115 120125 Pro Val Ser Met Leu Thr Ile Leu Tyr Gly Tyr Arg Trp Pro Leu Pro 130135 140 Ser Lys Leu Cys Ala Val Trp Ile Tyr Leu Asp Val Leu Phe Ser Thr145 150 155 160 Ala Ser Ile Met His Leu Cys Ala Ile Ser Leu Asp Arg TyrVal Ala 165 170 175 Ile Gln Asn Pro Ile His His Ser Arg Phe Asn Ser ArgThr Lys Ala 180 185 190 Phe Leu Lys Ile Ile Ala Val Trp Thr Ile Ser ValGly Ile Ser Met 195 200 205 Pro Ile Pro Val Phe Gly Leu Gln Asp Asp SerLys Val Phe Lys Glu 210 215 220 Gly Ser Cys Leu Leu Ala Asp Asp Asn PheVal Leu Ile Gly Ser Phe 225 230 235 240 Val Ser Phe Phe Ile Pro Leu ThrIle Met Val Ile Thr Tyr Phe Leu 245 250 255 Thr Ile Lys Ser Leu Gln LysGlu Ala Thr Leu Cys Val Ser Asp Leu 260 265 270 Gly Thr Arg Ala Lys LeuAla Ser Phe Ser Phe Leu Pro Gln Ser Ser 275 280 285 Leu Ser Ser Glu LysLeu Phe Gln Arg Ser Ile His Arg Glu Pro Gly 290 295 300 Ser Tyr Thr GlyArg Arg Thr Met Gln Ser Ile Ser Asn Glu Gln Lys 305 310 315 320 Ala CysLys Val Leu Gly Ile Val Phe Phe Leu Phe Val Val Met Trp 325 330 335 CysPro Phe Phe Ile Thr Asn Ile Met Ala Val Ile Cys Lys Glu Ser 340 345 350Cys Asn Glu Asp Val Ile Gly Ala Leu Leu Asn Val Phe Val Trp Ile 355 360365 Gly Tyr Leu Ser Ser Ala Val Asn Pro Leu Val Tyr Thr Leu Phe Asn 370375 380 Lys Thr Tyr Arg Ser Ala Phe Ser Arg Tyr Ile Gln Cys Gln Tyr Lys385 390 395 400 Glu Asn Lys Lys Pro Leu Gln Leu Ile Leu Val Asn Thr IlePro Ala 405 410 415 Leu Ala Tyr Lys Ser Ser Gln Leu Gln Met Gly Gln LysLys Ser Lys 420 425 430 Gln Asp Ala Lys Thr Thr Asp Asn Asp Cys Ser MetVal Ala Leu Gly 435 440 445 Lys Gln His Ser Glu Glu Ala Ser Lys Asp AsnSer Asp Gly Val Asn 450 455 460 Glu Lys Val Ser Cys Val 465 470 9 45 DNAArtificial Sequence Description of Artificial Sequence primer/probe 9tctcaccact ctccaaaagg acttggccat tcacctcctc ctttg 45 10 24 DNAArtificial Sequence Description of Artificial Sequence primer/probe 10tctattctgg aggcaccaag gaac 24 11 24 DNA Artificial Sequence Descriptionof Artificial Sequence primer/probe 11 tgttgatggg tcagataaag actt 24 1245 DNA Artificial Sequence Description of Artificial Sequenceprimer/probe 12 gtgatgcttg atgatgcact catcatctcg gcttgtcccc tggtg 45 1345 DNA Artificial Sequence Description of Artificial Sequenceprimer/probe 13 tagcagttcc tctgaggtca agttttgatc agaagagttt aagaa 45

What is claimed is:
 1. An isolated nucleic acid molecule encoding ahuman 5-HT_(1F) receptor.
 2. An isolated nucleic acid molecule of claim1, wherein the nucleic acid molecule is a DNA molecule.
 3. An isolatedDNA molecule of claim 2, wherein the DNA molecule is a cDNA moleculeencoding a human 5-HT_(1F) receptor.
 4. An isolated human 5-HT_(1F)receptor protein.
 5. A vector comprising the DNA molecule of claim
 2. 6.A plasmid comprising the vector of claim
 5. 7. A vector of claim 5adapted for expression in a bacterial cell which comprises theregulatory elements necessary for expression of the DNA in the bacterialcell so located relative to the DNA encoding the 5-HT_(1F) receptor asto permit expression thereof.
 8. A vector of claim 5 adapted forexpression in a yeast cell which comprises the regulatory elementsnecessary for expression of the DNA in the yeast cell so locatedrelative to the DNA encoding the 5-HT_(1F) receptor as to permitexpression thereof.
 9. A vector of claim 5 adapted for expression in amammalian cell which comprises the regulatory elements necessary forexpression of the DNA in the mammalian cell so located relative to theDNA encoding the 5-HT_(1F) receptor as to permit expression thereof. 10.A plasmid of claim 6 adapted for expression in a mammalian cell whichcomprises the regulatory elements necessary for expression of the DNA inthe mammalian cell so located relative to the DNA encoding the 5-HT_(1F)receptor as to permit expression thereof.
 11. A plasmid comprising thecDNA molecule of clam 3 adapted for expression in a mammalian cell whichcomprises the regulatory elements necessary for expression of the DNA inthe mammalian cell so located relative to the cDNA molecule as to permitexpression thereof, designated pMO5-h116a (ATCC Accession No. 75175).12. A mammalian cell comprising the plasmid of claim
 6. 13. An Ltk⁻ cellcomprising the plasmid of claim
 6. 14. An NIH3T3 cell comprising theplasmid of claim
 6. 15. An Ltk⁻ cell comprising the plasmid of claim 11,designated L-5-HT_(1F) (ATCC Accession No. CRL 10957).
 16. An NIL3T3cell comprising the plasmid of claim 11 designated N-5-HT_(1F) (ATCCAccession No. CRL 10956).
 17. A nucleic acid probe comprising a nucleicacid molecule of at least 15 nucleotides capable of specificallyhybridizing with a sequence included within the sequence of a nucleicacid molecule encoding a human 5-HT_(1F) receptor.
 18. The nucleic acidprobe of claim 17 wherein the nucleic acid is DNA.
 19. An antisenseoligonucleotide having a sequence capable of binding specifically to anmRNA molecule encoding a human 5-HT_(1F) receptor so as to preventtranslation of the mRNA molecule.
 20. An antisense oligonucleotidehaving a sequence capable of binding specifically to the cDNA moleculeof claim
 3. 21. An antisense oligonucleotide of claim 19 comprisingchemical analogues of nucleotides.
 22. An antibody directed to a human5-HT_(1F) receptor.
 23. A monoclonal antibody directed to an epitope ofa human 5-HT_(1F) receptor present on the surface of a cell and havingan amino acid sequence substantially the same as an amino acid sequencefor a cell surface epitope of the human 5-HT_(1F) receptor.
 24. Apharmaceutical composition comprising an amount of the oligonucleotideof claim 19 effective to reduce expression of a human 5-HT_(1F) receptorby passing through a cell membrane and binding specifically with mRNAencoding a human 5-HT_(1F) receptor in the cell so as to prevent itstranslation and a pharmaceutically acceptable hydrophobic carriercapable of passing through a cell membrane.
 25. A pharmaceuticalcomposition of claim 24, wherein the oligonucleotide is coupled to asubstance which inactivates mRNA.
 26. A pharmaceutical composition ofclaim 25, wherein the substance which inactivates mRNA is a ribozyme.27. A pharmaceutical composition of claim 24, wherein thepharmaceutically acceptable hydrophobic carrier capable of passingthrough a cell membrane comprises a structure which binds to a receptorspecific for a selected cell type and is thereby taken up by cells ofthe selected cell type.
 28. A pharmaceutical composition comprising anamount of a substance effective to alleviate the abnormalities resultingfrom overexpression of a human 5-HT_(1F) receptor and a pharmaceuticallyacceptable carrier.
 29. A pharmaceutical composition comprising anamount of a substance effective to alleviate abnormalities resultingfrom underexpressior of 5-HT_(1F) receptor and a pharmaceuticallyacceptable carrier.
 30. A pharmaceutical composition which comprises anamount of the antibody of claim 22 effective to block binding ofnaturally occurring ligands to the 5-HT_(1F) receptor and apharmaceutically acceptable carrier.
 31. A transgenic nonhuman mammalexpressing DNA encoding a human 5-HT_(1F) receptor.
 32. A transgenicnonhuman mammal expressing DNA encoding a human 5-HT_(1F) receptor somutated as to be incapable of normal receptor activity, and notexpressing native 5-HT_(1F) receptor.
 33. A transgenic nonhuman mammalwhose genome comprises antisense DNA complementary to DNA encoding ahuman 5-HT_(1F) receptor so placed as to be transcribed into antisensemRNA which is complementary to mRNA encoding a 5-HT_(1F) receptor andwhich hybridizes to mRNA encoding a 5-HT_(1F) receptor thereby reducingits translation.
 34. The transgenic nonhuman mammal of any of claims 31,32 or 33, wherein the DNA encoding a human 5-HT_(1F) receptoradditionally comprises an inducible promoter.
 35. The transgenicnonhuman mammal of any of claims 3:, 32 or 33, wherein the DNA encodinga human 5-HT_(1F) receptor additionally comprises tissue specificregulatory elements.
 36. A transgenic nonhuman mammal of any of claims31, 32 or 33, wherein the transgenic nonhuman mammal is a mouse.
 37. Amethod for determining whether a ligand not known to be capable ofbinding to a human 5-HT_(1F) receptor can bind to a human 5-HT_(1F)receptor which comprises contacting a mammalian cell comprising anisolated DNA molecule encoding a human 5-HT_(1F) receptor with theligand under conditions permitting binding of ligands known to bind to a5-HT_(1F) receptor, detecting the presence of any of the ligand bound toa human 5-HT_(1F) receptor, and thereby determining whether the ligandbinds to a human 5-HT_(1F) receptor.
 38. A method for determiningwhether a ligand not known to be capable of binding to the human5-HT_(1F) receptor can functionally activate receptor activity orprevent the action of a ligand which does so comprising contacting amammalian cell of claim 12 with the ligand under conditions permittingthe activation or blockade of a functional response, and detecting bymeans of a bioassay from the mammalian cell such as a second messengerresponse, and thereby determining whether the ligand activates orprevents the activation of the human 5-HT_(1 F) receptor functionaloutput.
 39. The method of claim 37 or 38 wherein the mammalian cell isnonneuronal in origin.
 40. A method of claim 39, wherein the mammaliancell nonneuronal in origin is an Ltk⁻ cell.
 41. A method of claim 39,wherein the mammalian cell nonneuronal in origin is an NIH3T3 cell. 42.A ligand detected by the method of claim 37 or
 38. 43. A method ofscreening drugs to identify drugs which specifically interact with, andbind to, the human 5-HT_(1F) receptor on the surface of a cell whichcomprises contacting a mammalian cell comprising an isolated DNAmolecule encoding a human 5-HT_(1F) receptor with a plurality of drugs,determining those drugs which bind to the mammalian cell, and therebyidentifying drugs which specifically interact with, and bind to, a human5-HT_(1F) receptor.
 44. A method of screening drugs to identify drugswhich interact with, and activate or block the activation of, the human5-HT_(1F) receptor on the surface of a cell which comprises contactingthe mammalian cell of claim 12 with a plurality of drugs, determiningthose drugs which activate or block the activation of the receptor inthe mammalian cell using a bioassay such as a second messenger assays,and thereby identifying drugs which specifically interact with, andactivate or block the activation of, a human 5-HT_(1F) receptor.
 45. Themethod of claim 43 or 44 wherein the mammalian cell is nonneuronal inorigin.
 46. The method of claim 45 wherein the mammalian cellnonneuronal in origin is an Ltk⁻ cell.
 47. The method of claim 45wherein the mammalian cell nonneuronal in origin is an NIH3T3 cell. 48.A pharmaceutical composition comprising a drug identified by the methodof claim 43 or 44 and a pharmaceutically acceptable carrier.
 49. Amethod of detecting expression of the 5-HT_(1F) receptor on the surfaceof a cell by detecting the presence of mRNA coding for a 5-HT_(1F)receptor which comprises obtaining total mRNA from the cell andcontacting the mRNA so obtained with the nucleic acid probe of claim 17under hybridizing conditions, detecting the presence of mRNA hybridizedto the probe, and thereby detecting the expression of the 5-HT_(1F)receptor by the cell.
 50. A method of treating abnormalities in asubject, wherein the abnormality is alleviated by the reduced expressionof a 5-HT_(1F) receptor which comprises administering to a subject aneffective amount of the pharmaceutical composition of claim 24 effectiveto reduce expression of the 5-HT_(1F) receptor by the subject.
 51. Amethod of treating an abnormal condition related to an excess of5-HT_(1F) receptor activity which comprises administering to a subjectan effective amount of the pharmaceutical composition of claim 24effective to reduce expression of the 5-HT_(1F) receptor in the subject.52. The method of claim 51 wherein the abnormal condition is dementia.53. The method of claim 51 wherein the abnormal condition Parkinson'sdisease.
 54. The method of claim 51 wherein the abnormal condition is afeeding disorder.
 55. The method of claim 51 wherein the abnormalcondition is pathological anxiety.
 56. The method of claim 51 whereinthe abnormal condition is schizophrenia.
 57. The method of claim 51wherein the abnormal condition is a migraine headache.
 58. A method oftreating abnormalities which are alleviated by reduction of expressionof a human 5-HT_(1F) receptor which comprises administering to a subjectan amount of the pharmaceutical composition of claim 30 effective toblock binding of naturally occurring ligands to the 5-HT_(1F) receptorand thereby alleviate abnormalities resulting from overexpression of ahuman 5-HT_(1F) receptor.
 59. A method of treating an abnormal conditionrelated to an excess of 5-HT_(1F) receptor activity which comprisesadministering to a subject an amount of the pharmaceutical compositionof claim 30 effective to block binding of naturally occurring ligands tothe 5-HT_(1F) receptor and thereby alleviate the abnormal condition. 60.The method of claim 59 wherein the abnormal condition is dementia. 61.The method of claim 59 wherein the abnormal condition is Parkinson'sdisease.
 62. The method of claim 59 wherein the abnormal condition is afeeding disorder.
 63. The method of claim 59 wherein the abnormalcondition is a pathological anxiety.
 64. The method of claim 59 whereinthe abnormal condition is schizophrenia.
 65. The method of claim 59wherein the abnormal condition is a migraine headache.
 66. A method ofdetecting the presence of a human 5-HT_(1F) receptor on the surface of acell which comprises contacting the cell with the antibody of claim 22under conditions permitting binding of the antibody to the receptor,detecting the presence of the antibody bound to the cell, and therebydetecting the presence of a human 5-HT_(1F) receptor on the surface ofthe cell.
 67. A method of determining the physiological effects ofexpressing varying levels of human 5-HT_(1F) receptors which comprisesproducing a transgenic nonhuman mammal whose levels of human S-HT_(1F)receptor expression are varied by use of an inducible promoter whichregulates human 5-HT_(1F) receptor expression.
 68. A method ofdetermining the physiological effects of expressing varying levels ofhuman 5-HT_(1F) receptors which comprises producing a panel oftransgenic nonhuman mammals each expressing a different amount of human5-HT_(1F) receptor.
 69. A method for identifying a substance capable ofalleviating the abnormalities resulting from overexpression of a human5-HT_(1F) receptor comprising administering a substance to thetransgenic nonhuman mammal of claim 31 and determining whether thesubstance alleviates the physical and behavioral abnormalities displayedby the transgenic nonhuman mammal as a result of overexpression of ahuman 5-HT_(1F) receptor.
 70. A method for treating the abnormalitiesresulting from overexpression of a human 5-HT_(1F) receptor comprisesadministering to a subject an amount of the pharmaceutical compositionof claim 28 effective to alleviate the abnormalities resulting fromoverexpression of a human 5-HT_(1F) receptor.
 71. A method foridentifying a substance capable of alleviating the abnormalitiesresulting from underexpression of a human 5-HT_(1F) receptor comprisingadministering the substance to the transgenic nonhuman mammal of eitherof claims 32 or 33 and determining whether the substance alleviates thephysical and behavioral abnormalities displayed by the transgenicnonhuman mammal as a result of underexpression of a human 5-HT_(1F)receptor.
 72. A method for treating the abnormalities resulting fromunderexpression of a human 5-HT_(1F) receptor which comprisesadministering to a subject an amount of the pharmaceutical compositionof claim 29 effective to alleviate the abnormalities resulting fromunderexpression of a human 5-HT_(1F) receptor.
 73. A method fordiagnosing a predisposition to a disorder associated with the expressionof a specific human 5-HT_(1F) receptor allele which comprises: a.obtaining DNA of subjects suffering from the disorder; b. performing arestriction digest of the DNA with a panel of restriction enzymes; c.electrophoretically separating the resulting DNA fragments on a sizinggel; d. contacting the resulting gel with a nucleic acid probe capableof specifically hybridizing to DNA encoding a human 5-HT_(1F) receptorand labelled with a detectable marker; e. detecting labelled bands whichhave hybridized to the DNA encoding a human 5-HT_(1F) receptor labelledwith a detectable marker to create a unique band pattern specific to theDNA of subjects suffering from the disorder; f. preparing DNA obtainedfor diagnosis by steps a-e; and g. comparing the unique band patternspecific to the DNA of subjects suffering from the disorder from step eand the DNA obtained for diagnosis from step f to determine whether thepatterns are the same or different and to diagnose therebypredisposition to the disorder if the patterns are the same.
 74. Themethod of claim 73 wherein a disorder associated with the expression ofa specific human 5-HT_(1F) receptor allele is diagnosed.
 75. A method ofpreparing the isolated 5-HT_(1F) receptor of claim 4 which comprises: a.inducing cells to express 5-HT_(1F) receptor; b. recovering the receptorfrom the resulting cells; and c. purifying the receptor so recovered.76. A method of preparing the isolated 5-HT_(1F) receptor of claim 4which comprises: a. inserting nucleic acid encoding 5-HT_(1F) receptorin a suitable vector; b. inserting the resulting vector in a suitablehost cell; c. recovering the receptor produced by the resulting cell;and d. purifying the receptor so recovered.